JP3276398B2 - Ascorbate interference-resistant composition, test device, and method for assaying a predetermined analyte - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to compositions, test devices and methods for determining the presence or concentration of a given analyte in a test sample. More specifically, the present invention provides for an analyte, such as glucose or cholesterol, or an analyte that can interact with an oxidase by using an indicator reagent composition that effectively resists ascorbate interference. It relates to a new and improved method for assaying a biological sample, for example a liquid test sample, such as urine, plasma or serum, for a given analyte having an oxidizing activity, for example occult blood. The indicator reagent composition receives a detectable and measurable response when contacted with a test sample containing a predetermined analyte. The indicator reagent composition of the present invention provides a more accurate and sensitive assay for a given analyte by essentially eliminating the interference effects of ascorbate present in the test sample. Therefore,
Due to the improved sensitivity achieved by the indicator reagent compositions of the present invention, assays of test samples for low concentrations of a given analyte, such as assays of urine for occult blood or glucose, or serum or plasma for glucose. An improved method for the assay is provided.

[0002]

BACKGROUND OF THE INVENTION As used herein and hereinafter,
The expression "predetermined analyte" refers to (a) whether it is capable of interacting with an oxidase to produce hydrogen peroxide,
(B) Defined as a compound that either exhibits peroxidation activity. Similarly, as used herein and hereinafter, the expression "redox mediator" refers to one or more of the following that is capable of interacting with a given analyte to produce molecular oxygen. Defined as a compound. The molecular oxygen then oxidizes the oxidizable dye resulting in a detectable color change. The "redox mediator" can be a combination of an oxidase and an enzyme peroxidase, or, depending on the given analyte of interest, a hydroperoxide. As a result, assays for analytes that can interact with appropriate oxidases and peroxidases, such as glucose, or for a given analyte that has peroxidative activity, such as occult blood, i.e., hemoglobin, are The chromogenic interaction is such that the degree and intensity of color change of the oxidizable indicator dye correlates with the concentration of a given analyte in the test sample. Assays for a given analyte are particularly useful for detecting or quantifying low concentrations of an analyte in a body fluid sample, such as blood, urine, feces or gastrointestinal contents.

[0003] For example, glucose is the sugar most commonly found in urine. The presence of a detectable amount of glucose in urine is known as diabetes. Because diabetes can be benign or pathological,
The physician must distinguish between these two forms.

Diabetes can also occur when blood glucose levels are normal, because renal glucose reabsorption falls below normal levels, resulting in some glucose spilling into the urine. This is the cause. This is a benign condition, such that diabetes occurs after eating a large amount of food or in combination with emotional stress. However, (true) diabetes is a pathological condition and is a major cause of diabetes. Signs of (true) diabetes include a marked increase in blood glucose and increased urine output. The urine glucose content of diabetics can be as high as 10%, with a content of 2-5% being commonly found.

[0005] A variety of assays are available for assaying urine for glucose. The most commonly used assay is an enzymatic assay based on the interaction of glucose oxidase with glucose. The enzymatic glucose oxidase test for glucose, as applied to urine,
Specific for glucose. Other sugars such as lactose, fructose, galactose and pentose are not substrates for glucose oxidase and therefore
It is not detected or measured.

[0006] In a standard assay of test samples for glucose, glucose oxidase first converts glucose in the test sample to gluconic acid and hydrogen peroxide in the presence of oxygen. The peroxidase, also present in the assay, then catalyzes the interaction of hydrogen peroxide with an oxidizable dye compound similar to o-tolidine. The dye compound is usually essentially colorless in the reduced state, but upon oxidation, due to peroxidase-catalyzed interaction with hydrogen peroxide, for example, o-tolidine gives a blue color. Subject to color change. The degree and intensity of the color change
It is directly proportional to the amount of hydrogen peroxide produced by the conversion of glucose. The amount of hydrogen peroxide produced by the conversion of glucose is then correlated to the original glucose concentration in the urine sample. In practice, a test strip is immersed in a urine sample and the resulting color change of the test strip is
Colorless to 2.0 showing glucose concentration below 0.1%
Compare with a color chart ranging from blue to blue, which indicates a urine concentration of at least%.

[0007] Peroxidase is an enzyme that catalyzes the oxidation of various compounds, such as phenols and amines, with peroxides. In addition, there are certain compounds called pseudoperoxidases, which behave in a manner similar to peroxidase enzymes. According to it, pseudoperoxidase liberates oxygen from hydroperoxide and transfers the oxygen to certain receptor compounds. Thus, pseudoperoxidases are generally similar to enzymes in that they catalyze or participate in the interaction of peroxides with oxidizable compounds similar to oxidizable dye compounds. Pseudoperoxidase is also called a peroxidation active substance, and includes hemoglobin and its derivatives.

[0008] Thus, peroxidatively active substances such as hemoglobin, hemoglobin derivatives, erythrocytes, myoglobin or combinations thereof catalyze the interaction of hydroperoxides with oxidizable dyes. In such interactions, the peroxidatively active substance mimics the peroxidase enzyme and catalyzes or participates in the interaction of the oxidizable dye with the hydroperoxide. Oxygen liberated from the hydroperoxide by the peroxide active substance is transferred to an oxidizable dye. The resulting interaction results in a detectable response, e.g., a color change, where the intensity and extent of the response indicates the presence or concentration of the peroxide active.

For example, a low concentration of blood in urine is called "occult blood". Occult blood is detected by an assay for the peroxidatively active compound hemoglobin. Occult blood in urine, feces or vomiting is usually not visible to the naked eye, but detection of occult blood is important for the diagnosis of stomach, intestinal and ureteral bleeding. Bleeding is caused, for example, by tumors, ulcers or inflammation of the organ in question. Currently, most methods for measuring the presence of occult blood in a test sample are based on the pseudoperoxidase activity of hemoglobin or myoglobin.

[0010] The presence of occult blood in the urine is also a sign of kidney or ureter damage. Normally, no detectable amount of occult blood is present in urine, even with very sensitive chemical methods. The presence of blood in urine or feces is a symptom of various abnormal conditions, such as cancer. The presence of blood in the urine, as indicated by a positive test for occult blood, is often a sign of bleeding in the ureter. Free hemoglobin is present in urine due to kidney diseases, infections, neoplasms or trauma affecting parts of the ureter. Free hemoglobin in urine is
It may be a sign of transfusion side effects, hemolytic anemia or paroxysmal hemoglobinuria, or it may appear due to various poisonings or after severe burns.

Therefore, accurate and sensitive assays of blood, urine, and other test samples for a variety of predetermined analytes must be available to both the laboratory and the home. The assay must provide accurate detection and measurement of a given analyte so that a proper diagnosis can be made and proper medical procedures can be performed, monitored and maintained. In addition, the assay may be used in a dipping and reading format for the purpose of convenient, economical, qualitative or quantitative determination of a given analyte in blood, urine or other test samples. Seems to be advantageous.

Furthermore, any method of assaying for a particular analyte of interest in blood, urine or other test samples may involve competing chemical or physical interactions, such as the aforementioned analyte in a test sample. By using an indicator reagent composition that undergoes a color change as a result of the interaction with a given analyte, rather than as a result of preferential interaction with an ascorbate-like component, other than the analyte, It must produce accurate, reliable and reproducible results. In addition, a method for assaying a given analyte may be used in blood, urine or other test samples for the purpose of rapid, economical and accurate determination of the given analyte.
It would be advantageous if suitable for use as a dry phase reagent strip. In addition, the methods and compositions used to assay a given analyte must not adversely affect or interfere with other test reagent pads present on the multi-species reagent strip.

To determine whether a body fluid of an individual contains a clinically significant amount of a given analyte, and to monitor the progress of a medical procedure to determine the effectiveness of the procedure,
Simple, accurate and inexpensive detection assays for certain analytes, such as glucose and occult blood, have been developed. Furthermore, of several different assay methods developed for the detection or measurement of a given analyte in a test sample, methods based on dry-phase test strips that are immersed and read have proven to be particularly useful. However, the dry phase test strip method is easily automated and provides reproducible and accurate results.

Some test strips used to assay a given analyte may undergo a change that can interact with the given analyte and be detected or measured, eg, a color change. Some have a single test area consisting of a small square pad of a suitable carrier substrate impregnated with the indicator reagent composition. Other test strips are multi-species reagent strips, including one test zone as described above for the assay of a particular predetermined analyte, and several additional test zones on the same strip. Other clinically important components included above and present in the test sample can be assayed simultaneously. For any type of colorimetric test strip, a test for a given analyte in a test sample, for example, blood or urine, involves immersing the colorimetric test strip in the test sample and then immersing the test strip in the test area. This is easily accomplished by comparing the resulting color to a standardized color chart provided on the container of the colorimetric test strip. For example, glucose and occult blood tests are often included in multiplexed test strips to allow for urine sample screening during routine physical examinations, as early detection of excessive amounts of these urine components is important. I do.

The test strip method is the simplest and direct assay for the presence of a given analyte. For glucose assays, the test area incorporates an oxidizable indicator dye similar to 3,3 ', 5,5'-tetramethylbenzidine; glucose oxidase; and peroxidase. For occult blood assays, the test area incorporates an oxidizable indicator dye and hydroperoxide. In either assay, the test area is colored according to the interaction of a given analyte present in the test sample with the glucose oxidase-peroxidase pair or hydroperoxide to oxidize tetramethylbenzidine. Receive change. According to the above method, the concentration of a given analyte in a urine sample can be easily measured visually by comparing the color of the test strip with a color chart immediately after immersing the test strip in the test sample. But it is possible for one person.

However, if ascorbate or ascorbate ions are present in the test sample, they will have a significant interference effect on the above-described redox assay for a given analyte. The most common form of ascorbic acid typically belongs to vitamin C. This vitamin is a vital nutrient and is found in many naturally occurring foods, such as fruits and vegetables. Vitamin C
Can also be synthesized and therefore can be taken as a food additive or even in tablet form. The health benefits of vitamin C have been known for some time, and as a result, vitamin C has become a relatively popular nutrient. That is, vitamin C is a popular food additive and also a vitamin agent and other popular ingredients.

However, the human body generally only absorbs as much vitamin C as necessary to meet short-term needs. Normally, vitamins are not stored in the body, and excess vitamin C is typically excreted through the urinary system. As a result, vitamin C is commonly present in urine samples undergoing clinical assays.

Ascorbic acid is a reducing agent that can interfere with clinical assays by reducing the oxidized colored form of the indicator dye into a reduced, colorless form of the dye. However, ascorbic acid can be oxidized. Therefore, ascorbic acid cannot act as a reducing agent if it is oxidized before it can interact with the oxidized dye, and therefore cannot interfere with the assay of a given analyte. Become.

Both in the literature and in-house screening studies, it has been discovered that the inclusion of a particular metal ion complex in the indicator reagent composition helps to overcome the problem of ascorbic acid interference. However, metal ion complexes are capable of chemically oxidizing dyes or exhibiting peroxidase activity and exhibiting behavior similar to peroxidase enzymes or pseudoperoxidases, such as hydrogen peroxide or hydroperoxide. Can catalyze a color-forming reaction between the dye and the oxidizable dye. Thus, while the metal ion complex eliminates the ascorbic acid interference effect, the metal ion complex may cause false-positive assays.

Researchers have found that certain ferric ion complexes are:
It has been found that it substantially reduces false positive assay results due to any metal ion complex used to eliminate ascorbate interference. Ascorbic acid is oxidized by ferric ion or a ferric ion complex, so that the interference of ascorbic acid is eliminated. Representative publications showing the oxidation of ascorbic acid by ferric ions include:

E. Pelizetti et al .: "Kinetics and Mechani
sm of the Oxidation of AscorbicAcid by Tris (1,10-p
henanthroline) iron (III) and Its Derivatives in Aqu
eous Acidic Perchlorate Media ", Inorg. Chem., No. 1
Volume 5, pages 2898-2900 (1976). Here,
Ascorbic acid and tris (1 ', 10-phenanthroline) in aqueous perchloric acid over a pH range of 3.5
It reacts with iron (III) and the rate of oxidation decreases with increasing pH.

LS Vann: "A Rapid Micro Method for
Determination of Ascorbic Acidin Urine by Ferric
Reduction, Clin. Chem., Vol. 11, pp. 979-985 (1965).

MMT Khan et al .: "The Kinetics of the
Reaction of Iron (III) Chelatesof Aminopolycarboxy
lic Acids with Ascorbic Acid '', J. Am. Chem. Soc.
90, 3386-3389 (1968), and MMT
Khan et al., J. Am. Chem. Soc., Vol. 89, p. 7104 (1
967). Here, the kinetics of ascorbic acid oxidation in the presence of ferric and cupric chelates in the pH range from 1.8 to 3.45 are discussed.

GS Laurence et al .: “The Detection of a
Complex Intermediate in the Oxidation of Ascorbic
Acid by Ferric Ion ", J. Chem. Soc. Dalton Tran
s., 1667-1670 (1972).

WC Butts et al .: "Centrifugal Analyzer D
etermination of Ascorbate in Serum and Urine with
Fe ³⁺ / Ferrozine ”, Clin. Chem., Vol. 21, 1493-1497.
Page (1975).

L. Pekkarinen: "The Mechanism of the
Autoxidation of Ascorbic Acid Catalyzed by Iron S
alts in Citric Acid Solution ", Finn. Chem. Lett.
233-236 (1974).

M. Kimura et al .: "Kinetics and Mechanism
of the Oxidation of L-AscorbicAcid by Tris- (oxala
to) Cobaltate (III) and Tris- (1,10-phenanthroline) I
ron (III) Complexes in Aqueous Solution '', J. Che
m. Soc. Dalton Trans., pp. 423-427 (1982
Year).

AE Martell: "Chelates of Ascorbic A
cid, Formation and Catalytic Properties '', Ascorb
ic Acid Chemistry, Metabolism and Uses, Chapter 7: P.
A. Seib and BM Tolbert, Adv. In Chem. Series
(American Chemical Society, District of Columbia, Washington), 153-
178 pages (1982).

However, the above-mentioned oxidation of ascorbic acid based on ferric ion is used for assaying a predetermined analyte by eliminating the interference of ascorbic acid in a reaction in which an oxidase and a peroxidase are paired. If they do, they will show clear disadvantages. Upon oxidation of ascorbic acid, ferric ions are reduced to ferrous ions. Ferrous ion is a good reducing agent and reduces an oxidized indicator dye similar to tetramethylbenzidine (TMB) from its colored (oxidized) form to a colorless (reduced) form It is possible. Thus, even though ferric ion eliminates primary interference by ascorbic acid, ferrous ions can then cause secondary interference, which can cause low assay results due to errors.

[0030]

Accordingly, a simple, accurate and reliable method for assaying test samples for low levels of a given analyte without primary or secondary interference due to ascorbic acid is provided. Providing would seem to be very convenient. Current test strips for use with a given analyte include suitable oxidases,
An indicator reagent composition that incorporates a peroxidase enzyme and a metal ion complex to reduce primary interference of ascorbate is incorporated. While current test strips used to assay for a given analyte are stable and sensitive, current test strips still need improvement in the area of sensitivity. Thus, it would be a significant advance in the art of diagnostic assays if test strips were to be much more sensitive to a given analyte in a test sample. The work that led to the development of the compositions, test devices and methods of the present invention was directed toward achieving improvements in ascorbate resistance and sensitivity.

[0031]

SUMMARY OF THE INVENTION Surprisingly and surprisingly, the compositions and methods of the present invention include bromate ions, chlorate ions, perchlorate ions, chromate ions, and peroxides, hydrochlorides. By containing in the indicator reagent composition a primary oxidant of ascorbate and a metal ion or metal, an organic oxidizing agent similar to a peroxide or an N-halogen compound, and a co-oxidizing agent selected from the group consisting of combinations thereof are contained in the indicator reagent composition. Eliminate secondary interference caused by ionic complexes. To achieve the full advantage of the present invention, bromate ions are included as co-oxidants in the indicator reagent composition. The co-oxidant converts the reduced form of the metal ion, such as ferrous ion, to an oxidized form, such as ferric ion,
It has been found that the reduced form of the metal ion oxidizes such that it cannot be utilized for interaction with the oxidized form of the indicator dye.
Thus, the problem of reduced assay sensitivity due to metal ions or metal ion complexes contained in the indicator reagent composition is overcome.

According to the above, by providing a more accurate assay method in an easily usable form, for example, a test strip of a dipping and reading type, a person in charge of a laboratory can quantitatively determine a predetermined analyte. The assay can be performed to immediately obtain a reliable assay result.
In addition, the test strip method may be performed by one individual at home to more closely monitor the level of a given analyte, such as glucose or occult blood, in a test sample similar to blood or urine, or to be monitored by the individual. Monitoring the success of certain medical procedures.

As will be described more fully below, the method of the present invention involves the use of a suitable carrier incorporating the indicator reagent composition of the present invention containing an indicator dye, a redox mediator, a metal ion complex and a co-oxidant. The use of a test strip with a test pad containing a substrate allows for a rapid, accurate and reliable assay for a given analyte. Where a given analyte is capable of interacting with an oxidase, such as glucose, the redox mediator includes a suitable oxidase and peroxidase enzyme. If a given analyte exhibits peroxidative activity, such as occult blood, the redox mediator will include hydroperoxide. Co-oxidants include bromate ion, chlorate ion,
It is selected from the group consisting of perchlorate ions, chromate ions, and organic oxidants similar to peroxides, hydroperoxides or N-halogen compounds, and combinations thereof.

Prior to the present invention, indicator dyes; redox mediators; metal ion complexes; chlorate ions, bromate ions,
An indicator reagent composition comprising perchlorate ion, chromate ion, and a co-oxidant selected from the group consisting of an organic oxidant similar to peroxide, hydroperoxide or N-halogen compound, and a combination thereof. There has been no known method for assaying a test sample for a given analyte using a combined oxidation and reduction reaction. Dry phase test strips containing indicator dyes, redox mediators and metal ion complexes have been used previously, but dry phase test strips incorporating such compositions have been attributed to the metal ions of the metal ion complex. Tended to give erroneous assay results due to secondary interference. Thus, such an incorrect assay reduced the sensitivity of the test strip to a given analyte in the test sample. In contrast, the indicator reagent composition of the present invention essentially eliminates both primary interference by ascorbate and, surprisingly and surprisingly, secondary interference due to metal ion complexes. As a result, the improved indicator reagent composition increases the sensitivity of the assay, thereby providing a more accurate and reliable assay for a given analyte using redox-based chemistry. I do.

Several other attempts have been found in prior patents and publications to achieve the above goals of increased assay sensitivity and reduced ascorbate interference. For example, with respect to ascorbate interference in a glucose assay, the method uses an enzyme that interacts with ascorbate since the ascorbate is filtered from the test sample prior to contacting the test sample with the test reagent. There is a spread. This allows Dahl
Quist in Canadian Patent 844,564 disclosed a test device for a glucose assay having a porous zone for receiving a test sample. The sample receiving area is free of test reagents and is provided with an ion exchange material that absorbs ascorbate present in the test sample.

US Patent No. 4,168,2 to Danninger et al.
No. 05 describes incorporating ascorbate oxidase into a test reagent formulation and enzymatically oxidizing ascorbate present in the sample to dehydroascorbate, a compound that does not adversely affect the assay. Was done. Japanese Patent Publication No. 58-55757 of Fuji Organ Pharmaceutical Co., Ltd. discloses that a test sample is pretreated with a metal chelate having, for example, ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid as a ligand to remove ascorbate,
It has been disclosed to assay test samples for cholesterol, glucose or uric acid.

In US Pat. No. 3,411,887, Ku
The elimination of ascorbate interference with reagent compositions, which relies on enzymatically oxidizing substances, such as glucose oxidase, by using an ascorbate "capture reaction system" has been described. This “capture reaction system” utilizes a heavy metal ion having an intermediate oxidation-reduction potential between the redox indicator dye and ascorbate. Suitable heavy metal compounds cited by way of example include cobalt, iron, mercury and nickel. Other publications disclosing the complexation and oxidation of ascorbate with cobalt include Ann. Chim. App by G. Bragagnolo, which teaches the oxidation of ascorbic acid solutions by air in the presence of metallic cobalt.
licata., Vol. 31, pages 350-368 (1941). Similar activity for Co (NH ₃ ) ₆ Cl ₃ was also reported by T. Iwasaki in J. Chem. Soc.
Volume, pages 820-826 (1942).

US Patent No. 4,310,6 to Burkhardt et al.
No. 26 describes the use of ammonium cobalt (III) complexes to reduce ascorbate interference in assays for peroxide actives. Burkhardt et al.
Ammonium cobalt (III) complexes, such as Co (NH ₃
) Organic hydroperoxide and a suitable indicator with ₆ Cl _3, for example 3,3 ', discloses a composition that includes 5,5'-tetramethylbenzidine.

Copper ions have also been used to exclude ascorbate interference from the assay. For example, I. P
echt et al., `` The Copper-Poly-L-Histidine Complex: I.
TheEnvironmental Effect of the Polyelectrolyte on
the Oxidase Activity of Copper Ions ", J. Am. Che
m. Soc., Vol. 89, p. 1587 (1968), disclosed that oxygen and copper catalysts can be used to oxidize ascorbate. NA Vengerova et al., `` The Ascorbate-Oxidase
Activity of the Cu ⁺² -Poly-4-VinylpyridineComplex A
lkylated with Bromoacetic Acid '', Vysokomol.soye
d. Volume A13, Issue 11, pages 2509-2517 (1971)
(Translated by KA Allen) discloses a method for synthesizing carboxymethyl derivatives of poly-4-vinylpyridine, in which Cu (II) polymer complexes increase ascorbate oxidation activity involving only copper ions Taught that. Other references on the oxidation of ascorbate by Cu (II) include Z. Sun et al .: "Studies on F.
unctional Latices: Catalytic Effects of Histamine-
Containing Polymer-latex-copper (II) Complex on the
Oxidation of Ascorbic Acid, Macromolecules, Chapter
19, 984-987 (1986), and YI Skurl
ator et al .: “The Mechanism of Ascorbic Acid Oxidation
by Cu (II) -Poly-4-Vinylpyridine Complexes '', Eur.
Polymer J., Volume 15, pages 811-815 (1979).

US Patent No. 4,288,541 to Magers et al.
Discloses the use of a mercuric ion complex, such as mercuric sarcosinate, to confer ascorbate resistance to a glucose oxidase-based assay for glucose. In addition to the patents and publications mentioned above, the problem of ascorbate interference in glucose assays is discussed in H. Giffo
rd et al .: J. Am. Med. Assoc., Vol. 178, pp. 149-150 (1961); P. O'Gorman et al .: Brit. Med. J., 603.
606 pages (1960), R. Brandt et al .: Clin. Chem.
Acta, Vol. 51, pp. 103-104 (1974) R. Brandt et al .: Am. J. Clin. Pathol., Vol. 68, 592-
It is discussed on page 594 (1977).

Other methods of eliminating ascorbate interference in the analytical determination of a given analyte include, for example, West German Patent No. 27 07 628, which describes a pretreatment of a urine sample with an oxidizing agent for an assay. Figure 3 shows a urine analysis of the wet phase with prior removal of ascorbate. Oxidizing agents disclosed as useful are sodium iodate, sodium periodate, calcium hydrochloride, potassium triiodide, sodium hydrochloride, chloroamine and bromosuccinimide. Also, EP-A-0 370 056 describes the use of iodate in a diagnostic method to avoid interference by reducing agents such as ascorbic acid.

US Patent No. 4,5, Mayambala-Mwanika et al.
No. 87,220 disclosed the use of chelated ferric ions in assays for peroxide actives to eliminate ascorbate interference. Mayambala-Mwanika
States that a ferric chelate similar to the ferric chelate of N- (2-hydroxyethyl) ethylenediaminetetraacetic acid (Fe-HEDTA) eliminates ascorbate interference and provides a false positive test for peroxide active compounds. Disclosed not to occur.

Ismail et al., US Pat. No. 4,755,472
No. 1,4 to test for peroxide active substances
A stable test pad having a carrier substrate impregnated with diisopropylbenzene dihydroperoxide and a benzene indicator in a molar ratio of hydroperoxide to indicator of about 0.9 to 3.0 was disclosed. In order to impart ascorbate resistance, it is also possible to include a ferric chelate. The test pad is stable during storage and does not lead to false positive tests with respect to other test pads present on multi-specimen test strips, such as glucose test pads based on peroxidase / potassium iodide indicators.

In contrast to the prior art and in contrast to currently available commercial test pads, the compositions of the present invention
The method and apparatus comprises the steps of: providing a predetermined analyte in a test sample, wherein the predetermined analyte is either capable of interacting with an oxidase or exhibiting peroxidase activity; Shows increased sensitivity in assays for detection or concentration determination of. The method of the present invention comprises (a) effectively eliminating primary interference by ascorbate by containing a metal ion complex, and (b) bromate, chlorate, perchlorate, chromate,
And an organic oxidizing agent similar to a peroxide, a hydroperoxide or an N-halogen compound, and a co-oxidizing agent selected from the group consisting of a combination thereof to effectively suppress secondary interference caused by a metal ion complex. Uses an indicator reagent to be eliminated.

For example, bromate ion is used as a reagent for titration analysis in analytical chemistry. Bromate ions in acidic media are almost as strong oxidizing agents as permanganate ions. In the presence of a strong reducing agent, the bromate ion (BrO
₃ ^-) is a bromide ion (Br ^-. Is reduced to, however,
Bromate ion can oxidize bromide ion to bromine (Br ₂ ) as shown in the following formulas (1) and (2). ^{_{BrO 3 - + 6H + + 6e}} - → Br - + 3H 2 O (1) BrO 3 - + 5Br - + 6H + → 3Br 2 + 2H 2 O (2) IM Kolthoff et al Volumetric Analysis, Vol. 3, Ti
tration Methods: Oxidation-Reduction Reactions, Chapter
In Chapter 12, Interscience Publishers, New York, NY (1971), the first step (1) of the reaction forms bromide ions, which then react with excess bromate ions in a pH-dependent reaction sequence. To produce free bromine (2). From the formulas (1) and (2), it is clear that acidic conditions are required for the reaction to proceed. Similarly, chlorate ions (ClO ₃ ⁻ ) do not exhibit oxidizing properties in neutral or alkaline solutions, but chlorate (HCl) in acidic solutions.
It is known that the presence of O ₃ ) exhibits strong oxidizing properties.

The oxidation of reduced metal ions by bromate ions has also been described. For example, the oxidation of ferrous ion, ie iron (II), by bromate ion is described in JP Bir
k: "Mechanism of the Bromate Ion Oxidation of Aq
uoiron (II) ", Inorg. Chem., Vol. 12, pp. 2468-2472 (1973); J.P. Birk et al .:" Mechanism of the Reduction of Brom "
ate Ion by Cyano (bipyridyl) Iron (II) Complexes '',
Inorg. Chem., Vol. 17, pp. 1186-1191 (1978);
And SG Kozub: “Kinetics and Mechanisms of the Broma
te Oxidations of Substitution-Inert Iron (II) Com
plexes in Acidic Aqueous Solution '', Dissertation, Xe
rox University Microfilms, Ann Arbor (1975).

In each of the above specific publications,
The reaction between bromate and ferrous ions has been studied under very acidic conditions in perchloric acid. In addition, it is known that bromate ions do not oxidize ascorbic acid at appreciable rates in the essentially neutral pH range of 5-7.

US Patent Application No. 3 filed April 13, 1989 and commonly assigned to the assignee of the present invention.
No. 37,620 discloses copper containing a water-soluble polymer (I
I) It is described that the use of the complex eliminates ascorbate interference from clinical assays. In addition, co-oxidants are also included to oxidize copper (I) ions back to copper (II) ions after interaction with ascorbate. The co-oxidant may be peroxide or N
An organic oxidizing agent similar to a halogen compound, or an inorganic oxidizing agent similar to chromate, mercuric ion, thallium (III) ion, cerium (IV) ion, manganese (III) ion, bromate or iodate. There can be.
U.S. Patent Application No. 337,620 does not teach or suggest any metal ions other than copper (II) ions as oxidizing agents to eliminate ascorbate interference.

As will be more fully demonstrated below,
Bromate, chlorate, perchlorate, chromate, and peroxides, hydroperoxides or N
An organic oxidizing agent similar to a halogen compound, and a co-oxidizing agent selected from the group consisting of combinations thereof, ascorbin when contained in the indicator reagent composition of the present invention having an essentially neutral pH value; Oxidizing the reduced form of the metal ion included in the composition to eliminate primary interference by the acid salt;
Reverts to the oxidized form of metal ions. Such a result is surprising and important. This result is surprising with respect to the pH range when a co-oxidant similar to bromate or chromate ion oxidizes the metal, and this result was originally due to the elimination of primary interference by ascorbate. The metal ions themselves in the composition act as secondary interferents, and the reduced form of the metal ions interacts with the colored oxidized form of the indicator dye to reduce the dye to its colorless reduced form That is important. According to this, due to this secondary interference, the indicator dye does not appear to have a sufficient discoloration effect corresponding to the concentration of the predetermined analyte in the test sample. Thus, for a given analyte, a low assay result due to error is provided.

However, the compositions, methods and test devices of the present invention are capable of interacting with oxidases,
Or to provide an accurate assay for a given analyte exhibiting peroxidase activity. Surprisingly, the methods and compositions of the present invention essentially eliminate both primary and secondary interference due to ascorbic acid present in the test sample. As a result, according to the method of the present invention, a metal ion complex for eliminating primary interference of ascorbate in assaying blood, urine and other test samples for a given analyte with a dry phase test strip, Bromate ions, chlorate ions, perchlorate ions, chromate ions, and peroxides, hydroperoxides or N-halogens for eliminating secondary interference due to reduced forms of the metal ions of the metal ion complex. By using an indicator reagent composition comprising an organic oxidizing agent similar to a compound and a co-oxidizing agent selected from the group consisting of combinations thereof, novel and unexpected results are achieved.

In summary, the present invention relates to a method for preparing a test sample comprising:
(A) is capable of interacting with an oxidase;
Or (b) new and improved compositions, test devices and methods for determining the presence or concentration of a given analyte that either exhibit peroxidase activity. The device includes a test pad that includes a suitable carrier substrate that incorporates an indicator reagent composition that interacts with a predetermined analyte to produce a detectable response. For home use, the indicator reagent composition produces a visually detectable response. For laboratory applications, the indicator reagent composition is:
Produces a response that can be detected visually or with the aid of an instrument. The carrier substrate of the test pad includes a water-absorbing material, such as filter paper; a layer or film made of a non-water-absorbing material, such as a polymerized material; or a combination thereof.
The indicator reagent composition is incorporated into the carrier substrate, and the carrier substrate retains the indicator reagent composition throughout the carrier substrate while maintaining the permeability of the carrier substrate by a given analyte of the test sample.

More specifically, the present invention is directed to a method for assaying a blood, urine or other liquid test sample for a given analyte by using a new and improved indicator reagent composition. . The predetermined analyte is a compound capable of interacting with an oxidizing enzyme, such as glucose, cholesterol, uric acid, alcohol or triglycerides; or a compound exhibiting peroxidative activity, such as hemoglobin, ie, occult blood. (A) an indicator reagent similar to a redox indicator capable of undergoing a color change in response to a predetermined analyte; (b) a redox mediator containing an oxidase and either peroxidase or hydroperoxide (C) metal ion complexes; and (d) bromate, chlorate, perchlorate, chromate, and organic oxidants similar to peroxides, hydroperoxides or N-halogen compounds, and the like. The indicator reagent composition comprising a co-oxidizing agent selected from the group consisting of combinations effectively eliminates both primary interference caused by ascorbic acid and secondary interference caused by a metal ion complex. Has been demonstrated to exhibit increased sensitivity to analytes.

The metal ion complex contained in the indicator reagent composition effectively eliminates the primary interference of ascorbate present in the test sample by oxidizing the ascorbate. In this redox reaction, the metal ions of the metal ion complex are reduced. Then, the co-oxidizing agent similar to bromate ion contained in the composition oxidizes the reduced form of the metal ion, and consequently the reduced form of the metal ion converts the oxidized form of the indicator dye to its colorless reduced form And it cannot be used for reduction. Therefore, the color change resulting from the contact between the test pad incorporating the indicator reagent composition and the test sample is not a spurious but a true color change, and the color change is determined by the amount of a given analyte in the test sample. Can be accurately correlated.

According to an important feature of the present invention, a more accurate and reliable assay for a given analyte in a liquid test sample, since the metal ion complex eliminates the ascorbate ion interference. Is achieved, and
Before the reduced form of the metal ion can interact with the colored oxidized form of the indicator dye, sufficient color change occurs due to the co-oxidant oxidizing the reduced form of the metal ion to its oxidized form. Is detected. Utilizing the indicator reagent composition of the present invention in a clinical test method, a sufficient color change of the indicator reagent composition, which responds to a predetermined amount of an analyte in a test sample, is detected. Or a qualitative or quantitative assay for a given analyte in a test sample, such as glucose or hemoglobin, will be more accurate. The color change, and thus the assay, is not interfered with by the ascorbate ion and by the reduced form of the metal ion present in the metal ion complex.

Thus, an important aspect of the present invention is that for a given analyte that is either capable of interacting with an oxidase or exhibiting peroxidase activity, Provide a simple, accurate, and reproducible method for assaying blood, urine, and other test samples. This method utilizes an indicator reagent composition that eliminates interference with primary and secondary assays due to ascorbate, and thus achieves increased sensitivity for a given analyte.

The method for assaying blood, urine or other test liquids for a given analyte comprises an indicator dye; (a)
Redox mediators containing either oxidases and peroxidases or (b) hydroperoxides; metal ion complexes; bromate, chlorate, perchlorate, chromate, and peroxides, hydroperoxides Or an indicator reagent composition comprising an organic oxidizing agent similar to an N-halogen compound and a co-oxidizing agent selected from the group consisting of combinations thereof, wherein the primary interference of ascorbate and metal The secondary interference of the reduced form of the ionic complex is essentially eliminated.

When the indicator reagent composition comes into contact with the test sample, it interacts with a predetermined analyte in the test sample, undergoes a detectable or measurable color change, and receives a predetermined analyte in the test sample. Is clearly indicated. A composition of the invention,
The methods and test equipment provide a sensitive assay for low concentrations of a given analyte and provide accurate assay results for a given analyte.

The above and other objects, advantages and novel features of the present invention will become apparent from the following detailed description of preferred embodiments of the invention, taken in conjunction with the accompanying drawings.

According to the method of the present invention, an indicator dye; a redox mediator; a metal ion complex; bromate ion, chlorate ion, perchlorate ion, chromate ion, and peroxide, hydroperoxide or N- By using an indicator reagent composition comprising an organic oxidizing agent similar to a halogen compound and a co-oxidizing agent selected from the group consisting of a combination thereof, a predetermined analyte in blood, urine and other test samples can be analyzed. A qualitative or quantitative assay is performed for a substance, such as glucose, cholesterol, uric acid or occult blood. According to an important feature of the invention, the predetermined analyte is:
(A) a compound capable of interacting with an oxidase, such as glucose, cholesterol, alcohol,
Either uric acid or triglycerides, or (b) a compound exhibiting peroxidase activity, such as hemoglobin (ie occult blood) and myoglobin. Therefore,
The redox mediator of the indicator reagent composition includes,
Either (a) a suitable oxidase and peroxidase, or (b) hydroperoxide.

The indicator reagent composition of the present invention has a primary interference effect on ascorbic acid present in a test sample and a secondary interference caused by a metal ion complex contained in the indicator reagent composition and interacting with ascorbic acid. Eliminates action essentially. Thus, the indicator dye undergoes a sufficient color change upon contact with a test sample containing the predetermined analyte, depending on the interaction of the predetermined analyte with the redox mediator.

The metal ion complex is included in the indicator reagent composition to oxidize ascorbic acid present in the test sample,
Therefore, it also eliminates the primary interference of ascorbate. U.S. Pat. No. 4,587,2 to Mayambala-Mwanika et al.
No. 20 discloses that certain ferric ion chelates are used to substantially eliminate primary interference of ascorbate. However, nevertheless, problems still exist with current methods and devices for assaying for a given analyte that can interact with an oxidase or exhibit peroxidase activity. For example, during the oxidation of ascorbic acid, the metal ions of the metal ion complex are reduced (eg, ferric ion chelates are reduced to ferrous ion chelates). Ferrous ions interact with the oxidized, or colored, form of the indicator dye,
It is a reducing agent that is powerful enough to reduce the oxidized form of the dye to its reduced, or colorless, form. Therefore,
Because some of the color changes are overturned, low test results due to errors are produced.

Surprisingly and surprisingly, by including in the indicator reagent composition a suitable co-oxidant, such as a bromate ion, the ferrous ion is capable of interacting with the oxidized form of the indicator dye. It has been found that it is oxidized to ferric ions, thereby preventing the interfering interaction between the ferrous ions and the oxidized indicator dye. As a result, the color change resulting from the redox mediator-mediated interaction between a given analyte and the indicator dye will be sharper. Accordingly, the accuracy and sensitivity of the assay for low concentrations of a given analyte is increased.

The improved accuracy and increased sensitivity to low levels of a given analyte obtained by the method of the present invention are particularly useful for urine assays for glucose or occult blood. A commercially useful urine assay for a given analyte comprises a stable indicator reagent composition, is sensitive, and preferably has a primary interference with ascorbic acid and, preferably, from ascorbic acid or from a test sample. Are resistant to secondary interference caused by the exclusion of ascorbic acid.

As discussed earlier, ascorbate and ascorbate ions are common interferents in diagnostic tests based on redox indicator dyes. Ascorbic acid interference in urine assays for glucose and occult blood is well known in the art and is preferably eliminated. Ascorbic acid interferes with the oxidation of the indicator dye, so that ascorbic acid in the test sample produces an apparently negative result for a given analyte. Therefore,
"Ascorbate resistance" is defined as negligible interference with the color change of the indicator dye when a urine sample contains as much as 200 mg (milligrams) of ascorbic acid per deciliter (dl) of sample.

Eliminating ascorbate interference in assays for a given analyte means that the detection of a low concentration of a given analyte in a test sample has a disease or injury that must be further tested. It is clinically important because the status can be displayed. As discussed above, and as discussed more fully below, the methods and test devices of the present invention accurately assay for low concentrations of a given analyte in a test sample. The methods and test devices of the present invention use an indicator reagent composition that undergoes a color change in response to only the concentration of a given analyte in a test sample, thereby providing a sensitive and reliable assay for the given analyte. Achieve a rich test. Surprisingly and surprisingly, the inclusion of a suitable co-oxidant in the indicator reagent composition further comprising an indicator dye, a redox mediator and a metal ion complex can reduce primary interference due to ascorbate ions. It has been found that by substantially eliminating the effects and second-order interference effects due to metal ions present in reduced form, the accuracy and sensitivity of the assay for a given analyte is substantially increased.

The methods and test devices of the present invention can also be used to determine the presence or quantitative concentration of a given analyte in plasma or serum, urine, feces and gastrointestinal contents, and more generally, many other biological fluids. It will be clear that the method can also be used to determine the concentration of a predetermined analyte which is a biological semisolid. Generally, any aqueous test sample or test sample that is soluble in aqueous solvent can be assayed. To take full advantage of the present invention, it is possible to use the compositions of the present invention as dry phase test strips to interact with oxidases in urine or other test samples, or to use peroxidase. The presence or concentration of a given analyte that is either capable of exhibiting activity is determined.

Since the indicator dye is oxidized and undergoes a color change in accordance with the amount of the peroxide active substance present in the test sample, the method and the test device using the composition of the present invention can be used for a predetermined analysis. Provides a more accurate, reliable, and clinically meaningful test for an object. The extent and intensity of the color change is attenuated due to the presence of ascorbic acid or the presence of reduced metal ions, both of which reduce the colored oxidized form of the indicator dye to its colorless reduced form. There is no such thing. Therefore, the sensitivity of the assay is increased,
A rapid, accurate, and reliable assay method for a given analyte that can be performed at home or in a laboratory to obtain essentially rapid assay results is completed.

The process of the present invention may interact with an oxidase to produce hydrogen peroxide and then interact with peroxidase to release oxygen or interact with hydroperoxide to release oxygen. Take advantage of the ability of a given analyte to either be released. In each case, oxygen then oxidizes the indicator dye. Oxidation of the indicator dye results in a color change of the indicator reagent composition, and the extent and intensity of the color change is directly proportional to the concentration of a given analyte in the test sample. Therefore, the indicator reagent composition of the present invention contains a redox mediator, ie, (a) an oxidase and a peroxidase or (b) a hydroperoxide, and an indicator dye, wherein the indicator dye is It undergoes a color change upon its conversion to its oxidized form by oxygen formed by the interaction of the vehicle with a given analyte present in the test sample.

The indicator reagent composition also contains a compound for eliminating primary interference of ascorbate from an assay for a given analyte. According to the present invention, the component included to eliminate ascorbate interference is preferably a metal ion complex. However, as will be discussed more fully below, it is also possible to use a salt of a water-soluble metal ion to eliminate the primary interference of ascorbate. The metal ion exists in its oxidized form, eg, ferric ion, and is capable of interacting with and oxidizing ascorbate to eliminate ascorbic acid interference. As a result, the metal ions are reduced to their reduced form, ie, ferrous ions. However, ferrous ions can interact with the colored oxidized form of the indicator dye to again form ferric ions and a colorless reduced form of the indicator dye. Thus, some of the color changes are overturned, resulting in low assay results due to errors.

As a result, and in accordance with an important feature of the present invention, the indicator reagent composition comprises bromate ion (BrO ₃ ⁻ ),
Chlorate ion (ClO ₃ ⁻ ), perchlorate ion (Cl
O ₄ ⁻ ), cumuloate ion (CrO ₄ ⁻² ), and a co-oxidant selected from the group consisting of peroxides, hydroperoxides, organic oxidants similar to N-halogen compounds, and combinations thereof. Effectively and quickly reoxidizes ferrous ions to ferric ions, thereby preventing the interaction of ferrous ions with the oxidized form of the indicator dye. Thus, sufficient color changes are detected and measured to provide an accurate and reliable assay for a given analyte.

As discussed more fully below,
The indicator reagent composition of the present invention can contain a buffer compound to adjust the pH to a range of about 5 to about 7. Within this pH range, the color change resulting from the oxidation of the indicator dye is the sharpest and distinguishable. However, at this slightly acidic or essentially neutral pH, the bromate, chlorate or perchlorate ions interact with the reduced metal ions to regenerate the oxidized form of the metal ions, and The formation of harmless reduction products similar to bromide ions is unexpected. The prior art teaches that bromate, chlorate and perchlorate ions act as oxidizing agents in very acidic media, for example media with a pH below about 4.

The following imbalanced reaction illustrates and summarizes the interactions that occur when a test sample containing a given analyte is contacted with a test device containing the indicator reagent composition of the present invention. . For illustrative purposes, the predetermined analyte is glucose, and the metal ion complex is N- (2-hydroxyethyl) ethylenediaminetetraacetic acid [Fe (III) -HED
TA], the ferric ion chelate and co-oxidant are bromate ions. The term "Ind.dye" refers to the indicator dye, "o
The terms "x" and "red" refer to the oxidized and reduced forms of the dye, respectively. Fe (II) -HEDTA is Fe (III)
) -Reduced form of HEDTA, or ferrous form. Glucose + Oxygen (O ₂₎ → hydrogen peroxide (H ₂ O ₂₎ + gluconate (3) Peroxidase H2O2 + Ind.dye (red) (colorless) → Ind.dye (ox) (colored) + H ₂ O (4) ascorbic Acid + Ind.dye (ox) (colored) → Dehydroascorbate + Ind.dye (red) (colorless) (5) Fe (III) -HEDTA + ascorbic acid → Fe (II) -HEDTA + dehydroascorbate (6) Fe (II) -HEDTA + Ind.dye ( ox) ( colored) → Fe (III) -HEDTA + Ind.dye (red) ( colorless) (7) bromate ^{_{(BrO 3 -) + Fe (}} II) -HEDTA → bromide ( ^{Br -) + Fe (III)} -HEDTA (8)

Equations (3) and (4) exemplify a reaction equation relating to a chromogenic assay for glucose, and the degree and intensity of discoloration generated in equation (4) are proportional to the amount of glucose in the test sample. I do. Equation (5) shows ascorbic acid that interacts with the oxidized indicator dye to attenuate the color change and produce a false negative assay result or interfere with the assay. Equation (6) shows a ferric ion chelate, Fe (III) -HEDTA, that eliminates primary interference of ascorbate by oxidizing ascorbic acid to harmless dehydroascorbate. Formula (7) represents the reduced form of the metal ion complex [Fe (II) -HE
DTA] interacts with the oxidized form of the indicator dye to produce the reduced form of the indicator dye, thus indicating the secondary interference of ascorbate that occurs when the degree and intensity of the color change is attenuated. Formula (8) represents the reduced form of the metal ion complex [Fe (II)
[HEDTA] is a bromate ion that interacts with ferrous ion to eliminate secondary interference of ascorbate. Equation (8) shows that bromate ions are reduced to harmless bromide ions and non-interfering Fe (III) -HEDTA is regenerated. This makes it possible to remove interference caused by ascorbate (primary interference) and ascorbate by including a metal ion complex and a suitable co-oxidant similar to bromate ion in the indicator reagent composition. Interference due to the added metal ion complex (secondary interference) is essentially eliminated.

The indicator dye contained in the indicator reagent composition is:
The indicator dye is capable of undergoing a detectable response, preferably a chromogenic response, in the presence of a predetermined analyte and a redox mediator, such as an oxidase-peroxidase pair or hydroperoxide. It is limited only in. Thus, the indicator dye is preferably a redox indicator that undergoes a color change upon its conversion from its reduced form to its oxidized form by the interaction of a given analyte with a redox mediator. The indicator dye must be stable enough that both a given analyte and redox mediator can be present before a color change occurs. In order to fully exploit the advantages of the present invention, the indicator dye undergoes a color change due to a variety of detectable and measurable colors and intensities, and the extent and intensity of the color change is determined by the amount in the test sample. It is possible to correlate with a predetermined analyte concentration.

A number of indicator dyes are suitable for use in the compositions of the present invention and are generally relatively easily oxidized to yield oxidation products that exhibit rich colors. Suitable classes of indicator dyes include, but are not limited to, benzidine-type indicator compounds and heterocyclic azine indicator compounds. Examples of heterocyclic azine indicator compounds include bis- (N-ethylquinol-2-one) -azine and (N-methylbenzthiazol-2-one)-(1
-Ethyl-3-phenyl-5-methyltriazole-2
-On) -azine, but is not limited to these. Examples of the benzidine type indicator compound include benzidine, o-tolidine, 3,3 ′, 5,5′-tetra (lower alkyl) benzidine, o-dianisidine,
There are, but are not limited to, 7-diaminofluorene and mixtures thereof, and other suitable indicator dyes. The expression "lower alkyl", as used above, is defined as an alkyl group having from 1 to about 6 carbon atoms and includes methyl, ethyl, n-propyl, isopropyl, and various butyl, pentyl and hexyl isomers. The body is included in this. To make full use of the advantages of the present invention, 3,3 ', 5,5'-tetramethylbenzidine (T
MB) is included in the indicator reagent composition.

Generally, the indicator dye is present in the indicator reagent composition at a concentration of about 5 to about 60 mM (mmol, ie, millimoles of indicator dye per liter of indicator reagent composition), preferably about 20 to about 40 mM. Exists. It is understood that the amount of indicator dye in the indicator reagent composition can be less than about 5 mM or greater than about 60 mM, depending on the intensity of the color change that the individual indicator dye undergoes upon oxidation. Must. In general, the amount of indicator dye included in the indicator reagent composition is such that the indicator dye undergoes a detectable color change for a qualitative assay and the amount of test dye in a test sample for a quantitative assay. Is limited only in that it undergoes a measurable color change proportional to the amount of a given analyte.

According to another important feature of the present invention,
The indicator reagent composition also contains a redox mediator. When the predetermined analyte is a compound capable of interacting with an oxidase, as the redox mediator, (a) an oxidase specifically interacting with the predetermined analyte and (b) ) Contains peroxidase enzymes. If the given analyte is a compound that can exhibit peroxidase activity, it will include hydroperoxide as a redox mediator.

In assays for a given hemoglobin-like analyte that exhibits peroxidase activity, the given analyte is capable of interacting with a redox mediator, including hydroperoxide. As a result of this interaction, the indicator dye is oxidized and undergoes a color change proportional to the amount of a given analyte in the test sample. A given analyte catalyzes the oxidation of the indicator dye if the given analyte present in the test sample exhibits peroxidation activity, ie, a behavior similar to the enzyme peroxidase.

Therefore, the indicator reagent composition of the present invention
The hydroperoxide included as a redox mediator for a given analyte exhibiting peroxidative activity must be sufficiently stable so that free oxygen is not released in the absence of the given analyte. Further, the hydroperoxide is
During storage or after the indicator reagent composition has been incorporated into the carrier substrate of the test pad of the dry phase test strip, it must have a sufficiently low vapor pressure so that the hydroperoxide does not evaporate or sublime. In addition, the hydroperoxide must be sensitive enough to detect one part of a given hemoglobin-like analyte in one trillion parts of the test sample, for example, in a urine assay for occult blood.

Accordingly, the hydroperoxides useful in the indicator reagent compositions of the present invention are selected from a number of well-known hydroperoxides. Suitable hydroperoxides are
It is possible to interact with a given analyte in the presence of a suitable indicator dye to produce a detectable response, such as a color change, or a change in the amount of light absorbed or reflected by the test sample. . Organic hydroperoxides are preferred. Specific examples of suitable hydroperoxides include
Cumene hydroperoxide, t-butyl hydroperoxide, diisopropylbenzene hydroperoxide,
1-hydroxycyclohexane-1-hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide, paramenthane hydroperoxide, 1,4-
Diisopropylbenzene monohydroperoxide, p
-T-butylisopropylbenzene hydroperoxide, 2- (α-hydroperoxyisopropyl) -6
There are, but are not limited to, isopropyl naphthalene, tetralin hydroperoxide and combinations thereof. In urine assays for occult blood, 1,4-diisopropylbenzene dihydroperoxide (DBDH) is the preferred hydroperoxide because it is stable, sensitive and nonvolatile.

The concentration of hydroperoxide contained in the indicator reagent composition is about 5 to about 100 mM, preferably about 25 to about 100 mM.
It is in the range of about 75 mM. The specific amount of a particular hydroperoxide included in the indicator reagent composition will depend on the physicochemical properties of each hydroperoxide, such as volatility, stability, and sensitivity to a given analyte exhibiting peroxide activity. It is decided according to.

In assays for a given analyte, such as glucose or cholesterol, capable of interacting with an oxidase, the redox mediator may be:
There are (a) an oxidase that specifically interacts with a predetermined analyte and (b) peroxidase. In this test,
A given analyte and oxidase interact first to produce hydrogen peroxide, which then interacts with peroxidase to oxidize the indicator dye and change the color of the indicator reagent composition. Where the extent and sensitivity of the color change is correlated with the amount of a given analyte in the test sample. As a result, the method of assay for a given specific analyte can be designed by using a redox mediator that includes the appropriate oxidase and peroxidase.

From the foregoing, the oxidizing enzyme contained in the indicator reagent composition can selectively interact with the given analyte of interest, as opposed to other components in the test sample. For example, in an assay for glucose, glucose oxidase is included as an oxidase, and in an assay for cholesterol, cholesterol oxidase is included as an oxidase. However, in addition to glucose oxidase and cholesterol oxidase, other oxidases that interact with a specific analyte to generate an oxidizing ability to oxidize the indicator dye include uricase and aryl alcohol oxidase. Enzymes, L-gluconolactone oxidase, galactose oxidase, pyranose oxidase, L-sorbose oxidase, pyridoxine 4-oxidase, alcohol oxidase, L-2-hydroxyacid oxidase, pyruvate oxidase, oxalate oxidase Enzymes, glyoxylate oxidase, dihydroorotate oxidase, lanosterol oxidase, choline oxidase, glycolate oxidase, glycerin-3-phosphate oxidase, xanthine oxidase, sarcosine oxidase, N-methylamino acid oxidase, N ⁶ methyl lysine oxidase, 6-hydroxy -L-nicotine oxidase, 6-hydroxy-D-nicotine oxidase, nitroethane oxidase, sulfite oxidase, thiol oxidase, cytochrome c oxidase, pseudomonas cytochrome oxidase, ascorbic acid oxidase, o-aminophenol Oxidase and 3-hydroxyanthranilate oxidase, but are not limited to these. Such oxidases can be used in assays for various predetermined analytes, such as triglycerides, uric acid, glucose, cholesterol, and galactose.

[0084] The concentration of the oxidase in the indicator reagent composition is sufficient to interact with the total amount of a given analyte in the test sample. Thus, in general, the amount of oxidase included in the indicator reagent composition ranges from about 50 to about 1,000 units per milliliter (ml) of the indicator reagent composition, and preferably from about 50 to about 250 units. Range of units [where 1 unit is the pH that is appropriate for the test in question.
And at a temperature of about 30 ° C., the amount of oxidase that oxidizes 1.0 μM (micromoles) of substrate per minute]. The amount of oxidase in the indicator reagent composition may vary depending on the particular predetermined analyte of interest and the expected concentration of the particular predetermined analyte in the test sample, at a rate of about 1,000 per ml.
It should be understood that there can be more than zero units or less than 50 units.

Similarly, the amount of peroxidase in the indicator reagent composition is sufficient to interact with the total amount of hydrogen peroxide produced by the interaction of the oxidase with a given analyte. Therefore, in general, the amount of peroxidase contained in the indicator reagent composition depends on the indicator reagent composition 1
It is in the range of about 50 to about 1,000 units per ml, preferably in the range of about 50 to about 250 units [where 1
The unit is 1 mg for 20 seconds at pH 6.0 and 20 ° C.
(Milligrams) of purprogalin is defined as the amount formed from pyrogallol]. The amount of peroxidase may be 1 ml, depending on the specific analyte of interest and the expected concentration of the specific analyte in the test sample.
It should be understood that there can be more than 1,000 units per unit, or less than 50 units per unit.

In addition, in addition to the indicator dye and redox mediator, the indicator reagent composition also contains a metal ion complex to impart ascorbate resistance to the assay. Generally, metal ion complexes promote the oxidation of ascorbate present in the test sample by the metal ion, thereby eliminating ascorbate interference. If ascorbic acid is not oxidized, ascorbic acid interacts with the colored oxidized form of the indicator dye to produce oxidized ascorbic acid,
And a colorless reduced form of the indicator dye. Therefore,
A low or false negative assay result for a given analyte will result in an error. Generally, the metal ion is included in the indicator reagent composition as a metal ion complex to increase the solubility of the metal ion and to help overcome the peroxidation activity inherent to the metal ion. However, it is also possible to include a water soluble salt of a metal ion in the indicator reagent composition.

According to an important feature of the present invention, essentially any metal ion capable of rapidly oxidizing ascorbic acid and eliminating ascorbate interference when complexed. However, it can be contained in the indicator reagent composition of the present invention. Therefore, the metal ions useful for the metal ion complex contained in the indicator reagent composition are ferric ion, cupric ion, mercuric ion, stannic ion, nickel (II) ion, manganese (III)
), Cadmium (II), zinc (II), molybdenum (V), chromium (IV), vanadium (III), cerium (IV), and combinations thereof. Furthermore, (III
Other metal ions having higher valence states can also be used as metal ions of the metal ion complex. In order to achieve the full advantage of the present invention, the metal ion present in the metal ion complex is ferric ion.

As described above, metal ions are complexed to increase the water solubility of the metal ions and to reduce the inherent peroxidative activity of the metal ions. However, the nature of the complexing agent is not specifically limited, for example, polycarboxyalkylamines similar to ethylenediaminetetraacetic acid or nitrilotriacetic acid; polycarboxylic acids similar to citric acid, oxalic acid, tartaric acid or gluconic acid or Polyhydroxy compounds similar to sorbitol; lignosulfonates; glucoheptonates; bis (dimethylglyoximato); salicylic acid complexes similar to bissalicylaldehyde ethylenediiminate; dithioic acid derivatives; 2,4-pentanedione derivatives; dipyridine derivatives; triethylenepyridineamine; cysteine, glycine or histidine-containing polypeptides; proline derivatives;
Thiocrown ethers similar to 22,25-octthiacyclooctosane; triphenylphosphine; or combinations thereof.

In particular, ferric ion complexes useful in the indicator reagent composition include polycarboxyalkylamine chelates of ferric ions, such as those of the following: N- (2-hydroxyethyl) Ethylenediaminetriacetic acid (Fe-HEDTA), ethylenediaminetetraacetic acid (Fe-EDTA), cyclohexylenediaminetetraacetic acid (Fe-CDTA), nitrilotriacetic acid (Fe
-NTA), iminodiacetic acid (Fe-IMDA), ethylenediaminediacetate tripropionic acid (Fe-EDDP, α
Form and β form), hydroxyethyliminodiacetic acid (F
e-HIMDA), diethylenetriaminepentaacetic acid (Fe
-DTPA), ethylene bis (oxyethylene nitrilo) tetraacetic acid (Fe-EGTA), N- (2-acetamido) iminodiacetic acid (Fe-ADA) or a combination thereof, but is not limited thereto. Absent. Polycarboxylalkylamine chelates of ferric ions are more fully described in U.S. Pat. No. 4,587,220 (incorporated by reference into the present invention). Other suitable ferric ion complexes include ferric citrate, ferric gluconate, ferric glucoheptonate, ferric bissalicylaldehyde ethylene diiminate and triethylene pyridine ferric amine. Preferred ferric ion complexes are Fe-HEDTA and Fe-EDTA. To fully demonstrate the advantages of the present invention, a ferric ion chelate of Fe-HEDTA is included in the indicator reagent composition of the present invention. However, it should be understood that the metal ion can also exist in uncomplexed form, for example, as a water-soluble salt. For example, and as will be more fully apparent below, mercuric nitrate has been used as an uncomplexed form of metal ion.

The metal ion complex is included in the indicator reagent composition in an amount ranging from about 0.5 to about 50 mM, preferably from about 1 to about 25 mM. When present in this amount, the metal ion complex essentially eliminates the primary interference of ascorbate in the assay of test samples containing up to about 200 mg / dl (milligram / deciliter). Furthermore, suitable metal ion complexes, such as Fe (III) -HEDTA, or suitable metal salts, similar to mercuric nitrate, are commercially available and can therefore be incorporated directly into the indicator reagent composition. You have to understand. Alternatively, in the production of the indicator reagent composition, e.g., a metal ion salt like ferric chloride hexahydrate _{(FeCl 3 · 6H 2 O)} , and about the same molar amount of N-(2-hydroxy A complexing agent similar to (ethyl) ethylenediaminetriacetic acid (HEDTA) is separately incorporated into the indicator reagent composition,
It is also possible to produce a metal ion complex in situ by forming a ferric ion chelate of Fe-HEDTA.

When a composition containing only an indicator dye, a redox mediator and a metal ion complex is used in a method for assaying a test sample for a given analyte, distinct disadvantages become apparent. As described above, a redox mediator is included in the composition to interact with a given analyte to oxidize the indicator dye. Metal ion complexes are included in the composition to eliminate primary interference of ascorbate from the assay. However, when the interference of ascorbate is eliminated by oxidation of ascorbate, the metal ions are reduced. This reduced form of the metal ion can interact with the colored oxidized form of the indicator dye resulting from the interaction of the given analyte with the redox mediator. The reduced form of the metal ion reduces the oxidized form of the indicator dye to its colorless reduced form, thereby attenuating the extent and intensity of the color change, resulting in lower test results for a given analyte due to error. .

Thus, the reduction of the oxidized indicator dye by the reduced form of the metal ion introduces significant constraints on the sensitivity of the dry phase test strip into the assay for a given analyte. However, as will be more fully described below, indicator dyes, redox mediators, metal ion complexes, bromate ions, chlorate ions, perchlorate ions, chromate ions, and peroxides, Dry phase test strips incorporating an organic oxidizing agent, such as hydroperoxide and N-halogen compounds, and a co-oxidizing agent selected from the group consisting of combinations thereof, can be used in assays for a given analyte as described above. Overcome the low test results due to errors observed as follows.

Surprisingly and surprisingly, by further including a suitable co-oxidant in the indicator reagent composition containing the indicator dye, the redox mediator and the metal ion complex, the reduced form of the metal ion is sufficiently enhanced. It has been found that the oxidized indicator dye is consequently not reduced to its colorless reduced form by the reduced form of the metal ion. Generally, bromate, chlorate or perchlorate, chromate, and peroxide, hydroperoxide or N 2
-Organic oxidants like halogen compounds and combinations thereof are suitable co-oxidants and are included in the indicator reagent composition of the present invention as water-soluble salts or water-soluble compounds.

Thus, for example, the bromate ion can be used as a counter cation in the indicator reagent composition such that (a) a sufficiently water-soluble bromate is formed when a water-soluble medium is desired. Bromates having sodium, lithium, calcium, magnesium, ammonium, alkylammonium, hydroxyalkylammonium, alkylphosphonium, or combinations thereof, wherein the alkyl or hydroxyalkyl group has 1 to about 6 carbon atoms. Or (b) counter cations such as quaternary alkylammonium, cyanine, pyridinium, picolinium, quinalquinium, quinolinium, such that a water-insoluble medium is desired to form a sufficiently water-insoluble bromate. , Quaternary alkylphosphonium or combinations thereof [Wherein the alkyl group has 6 or more carbon atoms] bromate having included as. Similar chlorate, perchlorate and chromate counter cations can also be used as co-oxidants, as long as they do not adversely affect the assay for a given analyte. To take full advantage of the present invention, bromate ions are included in the indicator reagent composition in the form of potassium bromate.

The co-oxidant can also be an organic oxidant such as a peroxide, hydroperoxide or N-halogen compound. Examples of peroxides and hydroperoxides include diisopropylbenzene hydroperoxide (DBDH), diisopropylbenzene monohydroperoxide, phenylcyclohexane hydroperoxide, p- (α-hydroperoxyisopropyl) benzoic acid, p- (bromoisopropyl) benzenehydro Peroxides and p- (α-hydroxy-α′-hydroperoxyisopropyl) benzene include, but are not limited to. DBDH is a suitable peroxide or hydroperoxide. A non-limiting example of an organic N-halogen compound is 1-halobenzotriazole, wherein the halogen atom is preferably a chlorine atom.

Generally, the co-oxidant is present in the indicator reagent composition in a concentration range of about 5 to about 100 mM, preferably about 20 to about 7 mM.
It is included in a concentration range of 0 mM. To take full advantage of the present invention, the co-oxidant is bromate ion and is included in the indicator reagent composition in a concentration range of about 40 to about 60 mM. Within the above concentration ranges, a sufficient amount of co-oxidant is present in the indicator reagent composition to oxidize the reduced form of the metal ion to its oxidized form.

The indicator reagent composition of the present invention containing a co-oxidizing agent allows for an accurate and sensitive assay for a given analyte. Metal ion complexes such as Fe (III) -H
EDTA allows rapid two-electron oxidation of ascorbic acid to eliminate ascorbic acid as an interferent in the assay. The oxidation of ascorbic acid is represented by the equilibrium reaction equation (10), where Fe (III) -HEDTA and Fe
(II) -HEDTA is a ferric and ferrous chelate of N- (2-hydroxyethyl) ethylenediaminetetraacetic acid (HEDTA), respectively. 2Fe (III) -HEDTA + ascorbic acid → 2Fe (II) -HEDTA + 2H + + dehydroascorbic acid salt (9) 6Fe (II) -HEDTA + BrO 3 - + 6H + → 6Fe (III) -HEDTA + Br - + 3H 2 O (10) 3 ascorbic acid + BrO ₃ ^- → 3 dehydroascorbic acid salt _{^{+ Br - + 3H 2 O (}} 11)

Surprisingly, after this, at a pH of about 5 to about 7, the bromate ion reduces the reduced metal ion of the metal ion complex to its lower valence state [Fe (II)-
HEDTA] to its higher valence state [Fe (II
I) -HEDTA], thereby preventing the redox interaction between the reduced metal ion and the oxidized indicator dye. This bromate ion and Fe (II) -HE
The interaction with DTA is shown in the equilibrium reaction equation (10).

The stoichiometry of equation (10) is determined in a qualitative titration experiment by adding a known amount of potassium bromate to a solution containing a known amount of ferrous ion buffered at pH 6.5. . O-phenanthroline is used to detect the remaining Fe (II) (ferrous) ion. Table 1 shows the results of the titration experiment. Generally, in a solution containing Fe (II), the activity of o-phenanthroline is maintained until the amount of potassium bromate added to the solution reaches its equivalence ratio. In each of the qualitative titrations, bromine (Br ₂ ) was 10 μm.
It is not detected even when using an analytical method that can detect a small amount of bromine as M (micromol). Accordingly, bromine under the conditions described above - reduction product of ferrous ion interactions, bromide - theoretically consequences to be ^(Br). Thus, from Table 1, the equivalent of potassium bromate is one sixth of its molecular weight, indicating a change in bromine from a + 5-valent oxidation state (BrO ₃ ⁻ ) to a −1-valent oxidation state (Br ⁻ ). It turns out that. According to this, from Table 1 and Equation (10),
To take full advantage of the present invention, the molar amount of bromate ions contained in the indicator reagent composition is at least one sixth of the molar amount of Fe (III) -HEDTA contained in the composition. . The overall reaction of Fe (III) -HEDTA, ascorbic acid and bromate ions is represented by the equilibrium reaction equation (1).
It is shown in 1).

[0100] Table 1 stoichiometric KBrO ₃ ^- Determination by Fe (II) -HEDTA oxide buffer: 0.5M citrate, pH6.5 Fe (II) initial concentration of -HEDTA: 10mM ──── ─────────────────────────────── experiment added KBrO ₃ ^- KBrO ₃ ^- concentration equivalent mole / o-phenanthroline number, Equimolar ratio of mM Fe (II) Color at the time of addition ─────────────────────────────────── 100 0 Red 2 0.33 0.2 Red 3 0.67 0.4 Red 4 1.0 0.6 Red 5 1.33 0.8 Red 6 1.67 1.0 Non-red 7 3.33 2.0 Non-red ──────────────────── ───────────────

These results indicate that assays for a given analyte are usually performed in an essentially neutral pH range of about 5 to about 7, while bromate ions are not available in low pH media. It is unexpected because it shows its oxidizing strength. In addition, the bromate ion does not oxidize the indicator dye and therefore does not result in false positive or falsely high test results, does not interfere with the interaction of a given analyte with the redox mediator,
Exhibits a sufficiently rapid reaction rate with the reduced form of the metal ion, acts in the neutral pH range required for enzymatic interaction, and has a strong coloration that interferes with the colorimetric response of the indicator reagent composition. It was found to be reduced to - different innocuous bromide ion and molecular bromine (Br ^₂₎ (Br).

According to another important feature of the invention,
It has been found that the concentration of the metal ion complex and the concentration of the co-oxidant in the indicator reagent composition affect the rate of ascorbic acid oxidation. For example, if ascorbic acid is 0.1%.
1 mM (mmol), 25 µm Fe (III) -HEDTA
M (micromolar), and the rate of ascorbic acid oxidation of aqueous solutions with variable amounts of potassium bromate and a buffer pH of 6.5 by measuring the decrease in absorbance at 265 nm (nanometers) did.

In particular, FIG. 1 shows that ascorbic acid contains 0.1%.
The absorbance at 265 nm (A) of a solution of pH 6.5 containing mM, 25 μM Fe (III) -HEDTA, and 0, 0.5, 1.5 or 10 mM potassium bromate.
₂₆₅ ) A series of plots against time was entered. A
A decrease of ₂₆₅ indicates that ascorbic acid has been oxidized. Thus, FIG. 1 shows the absence of potassium bromate (0 mM
KBrO ₃₎ under, A _{26 5} is Fe (III) -HED
It shows a gradual but measurable decrease due to the presence of TA. As the concentration of potassium bromate increases over the range of 0.5-10 mM, A
The rate of change of ₂₆₅ increases. Figure 2 is a plot of first-order rate constant for the oxidation step (k, sec ^-1) against concentration of potassium bromate indicates that given as a straight line that intersects the y-axis at 0.01 sec ^-1 ing. Under these conditions, reoxidation of Fe (II) to Fe (III) is apparently due to this ascorbate oxidation mechanism, since the rate constant for this reaction is proportional to the concentration of potassium bromate. It is a low speed stage. FIG. 3 shows that the potassium bromate level was fixed at 0.5 mM and Fe (III) -HED
As the concentration of TA was increased over the range of 0-50 μM, the rate of ascorbic acid oxidation was reduced to Fe (III) -HE
It shows that it is a function of the concentration of DTA. According to this, as the concentration of Fe (III) -HEDTA increases, the rate of ascorbic acid oxidation also increases.

Thus, and according to an important feature of the present invention, bromate ions, chlorate ions or perchlorate ions, chromate ions, and organic oxidants such as peroxides, hydroperoxides or N-halogen compounds Is included in the indicator reagent composition of the present invention as a co-oxidant for completing the oxidation-reduction cycle of the metal ion in the metal ion complex. Metal ion complexes similar to ferric ion chelates catalyze the oxidation of ascorbic acid, which is then regenerated by the co-oxidant. Thus, a co-oxidant increases the sensitivity of the assay for a given analyte. The improved indicator reagent composition also has low toxicity, is stable, has minimal side reactions,
It also has advantages such as high compatibility between the components of the indicator reagent composition. As will be more fully apparent below,
Bromate, chlorate or perchlorate, chromate, and peroxide, hydroperoxide or N
An indicator reagent composition containing an organic oxidizing agent similar to a halogen compound, at an essentially neutral pH at which the test sample is assayed for a given analyte, the reduced metal ion of the metal ion complex Eliminating secondary interference is surprising and surprising.

In addition to the essential components described above, the indicator reagent composition can also include a sufficient amount of a suitable buffer so that the indicator reagent composition does not become a result of a change in pH. Change the color of the indicator dye as a result of the interaction of the given analyte with the redox mediator. The test sample is often at a pH outside the desired pH range for the assay in question, so that a buffer is added to the test sample. Therefore, it is shown that the indicator reagent composition of the present invention can contain any of various known buffers. Buffers are particularly important in commercially acceptable dry phase test strips that can withstand the effects of urine pH and urine specific gravity. The function of the buffer is to maintain the indicator reagent composition at an appropriate pH to stabilize the indicator reagent composition and to cause the indicator dye to undergo the desired color change during the assay.

Buffers are usually included in the indicator reagent compositions of the present invention at concentrations ranging from 0 to about 600 mM, preferably from about 50 to about 400 mM, although in certain circumstances the concentration of the buffer may be reduced. It is possible to exceed or fall below this range. To take full advantage of the present invention, an optional buffer is present at a concentration ranging from about 150 to about 300 mM.
It has been found that the pH of the indicator reagent composition should generally be maintained at a slightly acidic to neutral pH for optimal assay results. Thus, a pH of about 5 to about 7, preferably about 6 to about 7, provides a clearer and more easily discernable color change to an assay for a given analyte. Surprisingly, bromate, chlorate, perchlorate, chromate, and organic oxidants such as peroxides, hydroperoxides or N-halogen compounds can reduce reduced metal ions to this slightly acidic nature. Optimum assay results with efficient oxidation in the neutral to neutral pH range, resulting in sufficient color change being detected and free of primary and secondary interferences due to reduced forms of ascorbic acid and metal ions, respectively. It was found to bring.

For example, the prior art discloses ferric chelates of N- (2-hydroxyethyl) ethylenediaminetriacetic acid [Fe
When a ferric ion complex similar to (III) -HEDTA] is contained in the indicator reagent composition, the indicator reagent composition is 6.5.
It is taught to buffer to a pH above, for example, a pH range of 6.7 to 7.0. Most preferably, 6.8-
Buffer pH to 6.9. This pH range provides the best balance of sensitivity, stability and ascorbic acid resistance when assaying urine samples that exhibit very different pH values and specific gravities. Surprisingly, bromate, chlorate, perchlorate, chromate, or peroxide,
The hydroperoxide or N-halogen compound, or a combination thereof, sufficiently oxidizes ferric ions resulting from the oxidation of ascorbic acid to ferrous ions at this essentially neutral pH.

According to the above, well-known buffers such as acetates, phthalates, borates, trichloroacetates, sulfosalicylates, phosphates, tartrates, citrates, succinates, maleates, 2,2-bis (hydroxymethyl) -2,2 ′, 2 ″ -nitrilotriethanol, 3,
3-dimethylglutaric acid, 3-N-morpholinopropanesulfonic acid (MOPS), malonic acid, 1,3-bis [tris (hydroxymethyl) methylamino] propane (bis-tris), tris (hydroxymethyl) aminomethane (Tris), tris (hydroxymethyl) aminomethane-maleic acid (tris-maleate), tris (hydroxymethyl) aminomethane-malonic acid (tris-malonate), 3-N- (trishydroxymethyl)
Methylamino-2-hydroxypropanesulfonic acid (T
APSO), 2-{[tris (hydroxymethyl) methyl] amino} ethanesulfonic acid (TES), 1,4-piperazinebis (ethanesulfonic acid) (PIPES),
-Morpholinoethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and other suitable buffers known in the art, or any combination thereof. A buffer may optionally be included in the indicator reagent composition of the present invention.

The indicator reagent composition of the present invention comprises an indicator dye, a redox mediator, a metal ion complex and bromate ion, chlorate ion, perchlorate ion, chromate ion, and peroxide, hydroperoxide or An improved method for determining the presence or concentration of a given analyte in a liquid test sample, comprising a co-oxidant selected from the group consisting of organic oxidants similar to N-halogen compounds and combinations thereof. Used in the method. The indicator reagent composition is stable prior to contact with the liquid test sample, and then interacts with a predetermined analyte in the test sample to allow identification and measurement either visually or by instrument. It has been proven to produce possible color changes. Furthermore, in addition to the essential components described above, the indicator reagent composition of the present invention includes an indicator for contacting and interacting with oxygen that is catalytically released in the interaction of a given analyte with a redox mediator. It is possible to include an optional component similar to a buffer in an amount sufficient to change the color of the dye. Thus, the unbuffered color change due to ascorbic acid and the reduced form of the metal ion accurately determines the presence or concentration of a given analyte in the test sample.

In addition to the essential components, the indicator reagent composition may contain, in addition to the buffer, any component that does not substantially change the properties and functions of the essential components and does not interfere with a predetermined analyte. Can also be included. For example, the indicator reagent composition can optionally contain a compound to improve wetting of the test pad of the test device by the test sample and to stabilize the oxidized indicator dye. Typically, the compound is an anionic or non-ionic surfactant. Anionic surfactants such as sodium dodecyl sulfate, sodium dioctyl sulfosuccinate and long carbon chain sulfates or sulfonates like sodium dodecylbenzenesulfonate are suitable surfactants. Non-ionic surfactants such as octoxynol, non-oxinol or ethoxylated fatty alcohols can also be included in the indicator reagent compositions of the present invention. The surfactant is present at a concentration of 0 to about 200 mM, preferably about 50 to about 1 mM.
A concentration of 50 mM is included in the indicator reagent composition.

The indicator reagent composition can include a polymeric material that improves the stability and uniformity of the discoloration of the test device. Suitable polymeric materials include, but are not limited to, polyvinylpyrrolidone, polyvinyl alcohol, gum arabic, gelatin, algin, carrageenan, casein, albumin, methylcellulose and similar natural and synthetic polymeric materials. Suitable polymeric materials are polyvinylpyrrolidone,
For example, PVP-K-30, a polyvinylpyrrolidone having a molecular weight of 40,000, commercially available from GAF, New York, NY. Generally, the polymeric material is included in the indicator reagent composition in an amount of 0 to about 5%, preferably about 1 to about 4% of the total amount of the indicator reagent composition.

In addition, an inert background dye can be included in the indicator reagent composition to improve color separation and differentiation of color changes in a chromogenic assay for a given analyte. A suitable background dye is ethyl orange, ie 4- (4-diethylaminophenylazo)
Benzenesulfonic acid, orange G, ie 4- (2-
Hydroxy- (sodium 7,9-disulfonate) -1
-Naphthylazo) benzene, disperse orange 11, 13 or 25, chalcmine orange, methyl orange, and orange (II), ie, 4- (2-hydroxy-1-naphthylazo) benzenesulfonic acid, or combinations thereof. However, the present invention is not limited to these. The background dye ranges from 0 to about 2 mM, preferably from about 0.1 to about 1.2.
Concentrations in the range of mM are included in the indicator reagent composition of the present invention.

Water is a carrier vehicle for the components included in the indicator reagent composition. However, since the solubility of certain components contained in the indicator reagent composition in water is limited, organic solvents such as methanol, ethanol, isopropyl alcohol, acetone, dimethylformamide,
1-Methoxy-2-propanol, dimethyl sulfoxide, acetonitrile, ethyl acetate and similar solvents can be included in the carrier vehicle. The selection of a suitable organic solvent or solvents to be included in addition to the water in the carrier vehicle of the indicator reagent composition is within the ability of those skilled in the art of designing diagnostic assays.

The amount of organic solvent present in the indicator reagent composition generally ranges from 0 to about 90%, preferably from about 10 to about 70% by weight of the carrier vehicle. Carrier vehicles comprising water and an organic solvent similar to ethanol or acetonitrile are particularly preferred, since the carrier substrate impregnated with the indicator reagent composition can be dried within a few minutes to five or six minutes. .

As mentioned above, the indicator reagent composition undergoes a color change upon contact with the test sample, revealing the presence of a given analyte. Further, the intensity and degree of the color change
Measuring the quantitative concentration of a given analyte in a test sample by comparing or correlating the color produced by the test sample with a color produced by a solution containing a known concentration of the given analyte. It is used for According to an important feature of the present invention, the indicator reagent composition of the present invention measures the amount of a given analyte in a test sample without using a color measuring instrument, such as a spectrophotometer or a colorimeter. It has been shown to provide a separate and differentiated color change that is sufficient to allow accurate and accurate determination. However, if desired, such color measuring instruments can be used to measure differences in color intensity and intensity between a test sample and a solution containing a known concentration of a given analyte. is there.

Thus, an assay for a given analyte using the indicator reagent composition of the present invention improves the accuracy and reliability of the assay and also increases the physician's confidence in the assay. In addition, due to the large number of assays performed on a given analyte at home by untrained patients as opposed to trained physicians or technicians in the laboratory, test samples in urine-like It is imperative to provide an accurate and reliable quantitative test method for a given analyte, such as glucose.

To demonstrate the novel and unexpected results achieved by the method of the present invention, an indicator dye, a redox mediator, a metal ion complex, a bromate ion, a chlorate ion, a perchlorate ion, An indicator reagent composition containing a chromate ion and a co-oxidant selected from the group consisting of a peroxide, a hydroperoxide or an organic oxidant similar to an N-halogen compound, and a combination thereof is subjected to a predetermined analysis. It was used for dry phase test strip assays on samples. Dry phase test strip assays using the indicator reagent compositions of the present invention are performed according to methods well known in the art.
Generally, the assay for a given analyte involves contacting whole blood, serum, plasma, urine, or other test sample with an analyte detection device (detector) having the indicator reagent composition. It is implemented by. The analyte detector can be immersed in a test sample, or the test sample can be applied drop by drop to the analyte detector. The resulting color change of the analyte detector proves the presence of a given analyte and, if so designed, the resulting color change is a standardized color chart table. It is possible to achieve a quantitative measurement of the concentration of a given analyte in a test sample as compared to.

Typically, the analyte detection device is a test strip impregnated with a reagent, such as a single pad test strip (to test only one type of analyte) or a multi-pad test strip. (To assay several analytes simultaneously). For any type of reagent-impregnated test strip, the test strip typically comprises a carrier strip or handle, usually made of hydrophobic plastic, and a hygroscopic or non-hygroscopic carrier substrate incorporating the indicator reagent. Test pad. Generally, a carrier substrate is an absorbent material that allows a test sample to travel through the carrier substrate and contact the indicator reagent composition in response to capillary action to produce a detectable or measurable color change.

The carrier substrate can be any substance capable of incorporating the chemical reagents necessary to carry out the assay in question, as long as the carrier substrate is substantially inert with respect to the chemical reagent. And is porous or absorbent for liquid test samples. The expression "carrier substrate" is insoluble in water and other physiological fluids,
And a hygroscopic or non-hygroscopic substrate that retains its structural integrity when in contact with water and other physiological fluids. Suitable hygroscopic substrates include filter paper, sponge materials, cellulose, wood, woven and nonwoven fabrics, and the like. Non-hygroscopic substrates include glass fibers, polymer films, and preformed or microporous membranes. Suitable carrier substrates include, in addition, hydrophilic inorganic powders such as silica gel, alumina,
Diatomaceous earth or the like; clay material; cloth; hydrophilic natural polymeric materials, especially cellulosic beads and cellulosic materials, especially like fibre-containing paper, such as filter paper or chromatographic paper; synthetic or modified naturally occurring materials Polymers such as cellulose acetate, polyvinyl chloride, polyacrylamide, polyacrylate, polyurethane, cross-linked dextran, agarose, and other such cross-linked and non-cross-linked hydrophilic polymers that are insoluble in water. Hydrophobic and non-absorbable materials are unsuitable for use as carrier substrates in the present invention. The carrier substrate can also consist of different chemical compositions or a mixture of chemical compositions. Substrates can also vary in smoothness and roughness, combined with hardness and softness. Most conveniently, the carrier substrate is made from a hygroscopic filter paper or a non-hygroscopic polymer film. But,
In all cases, the carrier substrate comprises a hydrophilic or absorbent material. The handle is usually formed from a hydrophobic material such as cellulose acetate, polyethylene terephthalate, polycarbonate or polystyrene.

When the test strip is designed to assay a given analyte in a test sample, the carrier substrate is impregnated with the test sample and the test pad of the test strip incorporating the indicator reagent composition. It can be any hygroscopic or non-hygroscopic material that allows it to be saturated. To fully utilize the advantages of the present invention in assays for a given analyte in a test sample, the carrier substrate is a cellulosic material, for example a hydrophilic, hygroscopic substrate such as paper, preferably filter paper. The filter paper has all of the properties required for the hygroscopic substrate of the present invention, and in addition has the advantages of abundant supply, advantageous economics, and a variety of suitable grades. Filter paper has been found to be quite sufficient for use as a substrate material for suspension and emplacement of the essential and all optional components contained in the indicator reagent composition.

To fully exploit the advantages of the present invention, the indicator reagent composition is incorporated into a suitable carrier substrate and used as a dry phase test strip for assaying for a given analyte in a test sample. The method of the present invention provides an economical, accurate and reliable assay for the presence or concentration of a given analyte in a test sample, which can be performed at home or in a laboratory. Furthermore, the method of the present invention allows the detection, identification and measurement of low concentrations of a given analyte in a test sample, thus making the assay more clinically useful.

In accordance with the method of the present invention, from about 50 to about 1 to perform assays on dry phase test strips for a given analyte, such as glucose, capable of interacting with an oxidase. 2,000 units of the appropriate oxidase, about 50 to about 1,000 units of the enzyme peroxidase, about 5 to about 60 mM indicator dye, 0.5 to about 50 mM
A metal ion complex of about 5 to about 100 mM, bromate ion, chlorate ion, perchlorate ion, chromate ion,
And a co-oxidant selected from the group consisting of peroxides, hydroperoxides, and organic oxidants similar to N-halogen compounds, and combinations thereof, a buffer of 0 to about 600 mM, such as a citrate buffer, An aqueous solution is first prepared containing 200 mM surfactant, 0 to about 5% of the polymeric material, and any other desired optional ingredients similar to background dyes or solvents. The aqueous solution is then adjusted to a pH of about 5 to about 7 using a suitable organic or inorganic acid, such as 1N hydrochloric acid. Further, a hygroscopic substrate, such as filter paper, is saturated with the aqueous solution by immersing a sheet or a cut piece of the filter paper in the aqueous solution or spraying the aqueous solution on it.

Thereafter, the aqueous solvent is removed by drying the saturated filter paper in an air oven at a temperature of about 40 to about 100 ° C. for about 20 minutes. After oven drying, the filter paper impregnated with the reagent is sized to an appropriate size, for example, about 0.25.
Cut into pads of cm x about 0.5 cm to about 0.5 cm x about 1.0 cm.

Similarly, to perform an assay on a dry phase test strip for a given analyte similar to occult blood that exhibits peroxidase activity, the redox medium is changed from oxidase and peroxidase to hydroperoxide. Thus, from about 5 to about 60 mM indicator dye, from 0.5 to
About 50 mM metal ion complex, about 5 to about 100 mM hydroperoxide, about 5 to about 100 mM co-oxidant, 0 to about 6
00 mM buffer, such as sodium citrate, 0 to about 5
An aqueous solution is first prepared containing 0% to about 200 mM of a polymeric material, 0 to about 200 mM of a surfactant, background dye or any other desired optional ingredient similar to a solvent. Then, after adjusting the pH to about 5 to about 7, the aqueous solution is incorporated into a carrier substrate, the carrier substrate is dried, and finally cut to size as discussed above.

In each case, the dried and cut reagent-impregnated filter paper is then secured to an opaque or transparent hydrophobic plastic handle using double-sided adhesive tape.
The resulting test strip is then immersed in a test sample, eg, a fresh, uncentrifuged urine sample, for a time sufficient for the test pad to become saturated with the sample. Predetermined time,
After waiting for about 15 to about 60 seconds, for example, the test strip is tested for response, either visually or by instrument. The color change of the test pad, if any, is an indication of the presence or concentration of a given analyte in the urine sample.

It should be understood that, in order to design a quantitative assay for a given analyte using the methods and compositions of the present invention, the size of the reagent pad, the strength of the solution containing the reagent, Determining the proper balance between the amount of test sample and the method of directing the test sample to the test strip, for example, by using a pipette rather than immersion, is within the purview of those skilled in the art of manufacturing test devices. It is enough to exist.

In many cases, the desired information is obtained by mere visual observation of the test strip. If more accurate information is required, a color chart containing color spots corresponding to various known concentrations of a given analyte may be produced for the particular indicator reagent composition used in the test strip. It is possible to The color developed on the test strip after contact with the test sample is then compared to the color spot on the color chart to determine the concentration of a given analyte in the test sample. If more accurate judgment is required,
Using a spectrophotometer or a colorimeter, it is possible to more precisely measure the degree of color change. In addition, assays with dry phase test strips provide a more reliable and accurate measure of the degree of color change and, consequently, the concentration of a given analyte in a test sample, especially at low concentrations, For example, at concentrations below 1 mg / dl, this can be made quantitative by using a spectrophotometric or colorimetric method as opposed to a visual method to measure more accurately. is there.

Thus, according to an important feature of the present invention, by incorporating the indicator reagent composition of the present invention into a suitable carrier substrate, the determination of the presence or concentration of a given analyte in a test sample can be determined by: It is shown that this can be achieved with a dry phase test strip. As discussed above, dry phase test strips used for assaying a given analyte in a test sample generally include a carrier substrate, including any absorbent substrate, which can be an indicator reagent. Allowing blood, urine or other test samples to penetrate quickly enough so that treatment with the composition and its incorporation can be readily accommodated and a reliable assay obtained relatively quickly; and It does not contaminate other test samples by extraction of the components contained in the carrier substrate with the test sample or by significantly altering the test sample such that subsequent assays are indeterminate, inaccurate and suspicious. Substrate.

[0129]

EXAMPLES In accordance with one embodiment of the present invention, the following dry phase test strips were prepared and subjected to a dry phase assay for glucose. Glucose is capable of interacting with glucose oxidase to produce hydrogen peroxide. Therefore, the following aqueous composition of Example 1 was prepared. The composition of Example 1 is then applied to a carrier substrate strip or sheet similar to a porous polyurethane film,
The coating was performed using a Meyer rod.

Examples Indicator Reagent Composition Example 1 Example 2 Component Concentration Concentration Tetramethylbenzidine (TMB) (indicating dye) 35 mM 35 mM Sodium citrate (buffer) 250 mM 250 mM Ferric chloride (metal ion) 5 mM-N -(2-Hydroxyethyl) ethylenediaminetetraacetic acid (complexing agent for metal ions) 5 mM-Peroxidase 1000 units / ml 1000 units / ml Potassium bromate 20 mM-Glucose oxidase 1000 units / ml 1000 units / ml Sodium dodecyl sulfate (interface Activator) 28mm 28mM hydrochloric acid (1N) Adjust pH to about 6.0 Adjust pH to 6.0

The layer of the indicator reagent composition on the polyurethane substrate was then dried for about 10 minutes in an oven at a temperature ranging from about 45 to about 60 ° C. In addition, a polyurethane substrate incorporating the dried indicator reagent composition is about 0.5 cm × about 0.1 cm.
The test pads were cut into 5 cm sized pads and contained a carrier substrate incorporating the indicator reagent composition of the present invention. Thereafter, the test pad was fixed to a hydrophobic plastic handle using a double-sided adhesive to form a dry phase test strip of the present invention.

It should further be understood that during the production of the polyurethane substrate, it is possible to incorporate the indicator dye directly into the polyurethane substrate. The indicator dye often has low solubility in water, which makes it difficult to dissolve the indicator dye in an aqueous solution. However, since the indicator dye often exhibits excellent solubility in an organic solvent, the indicator dye is dissolved in the organic solvent used in producing the polyurethane substrate.

To demonstrate the novel and unexpected results achieved using the method of the present invention, a dry phase test strip incorporating the indicator reagent composition of the present invention (Example 1) was prepared using the components of Example 1 , But incorporated the indicator reagent composition (Example 2), but without bromate ion, ferric chloride and N- (2-hydroxyethyl) ethylenediaminetetraacetic acid. Test strips incorporating the composition of Example 2 were used as controls to demonstrate the improved assay of the present invention, ie, provided by the indicator reagent composition of Example 1.

An individual dry phase test strip incorporating either the composition of Example 1 or the composition of Example 2 was prepared by
Comparisons were made in assays of standard solutions containing 100 mg / dl glucose and 0-200 mg / dl ascorbic acid. The test strips are immersed in a standard solution containing glucose and ascorbic acid, and approximately one minute after contact with this standard solution, Miles Inc., Elkhart, Indiana.
The reflectance at 660 nm (nanometers) of the test pads of the test strips was measured using an Advanced Research Rapid Scanner reflectance spectrometer from the Department of Diagnostics, Inc.
Reflectance measurements represent the reactivity of a solution containing a standardized amount of glucose with a particular dry phase test strip.

Table 2 shows the dry phase test strips containing the indicator reagent composition (Example 2) in the absence of bromate and metal ion complexes, and the test strips containing the composition of the present invention (Example 1). Is a summary of the reactivity of
Overall, from Table 2, it can be seen that assays performed using test strips incorporating the indicator reagent composition of Example 1 were substantially more effective than assays performed using test strips incorporating the composition of Example 2. It shows strong resistance to ascorbic acid.

Table 2 Improved ascorbate resistance of test strips containing metal ion complex and bromate ion ────────── Test sample Glucose concentration (mg / dl) Ascorbate concentration (mg / dl) ────────────────────── ───────────── A 100 0 B 100 50 C 100 100 D 100 200 ───────────────────────── ────────── Test sample Indicator reagent composition Example 1 Example 2 ──────────────────────────── ─────── A 100 ¹⁾ 100 ¹⁾ B> 30 30 C> 30 0 D> 300 0 ──────────────────────── ─────────── 1) Apparent concentration of glucose in standard test sample, unit : Mg / dl.

The test strips incorporating the indicator reagent composition of the present invention (Example 1) were evaluated for ascorbate resistance by comparison with a control strip incorporating the indicator reagent composition of Example 2. Each of the assays contains 0, 30 or 100 mg / dl glucose, or 100 mg / dl glucose and 0, 50, 1
The strips were dipped in a standard glucose solution containing either 00 or 200 mg / dl ascorbic acid.
Standard solutions containing 0, 30, or 100 mg / dl glucose and no ascorbic acid produced a normalized response to glucose. The responses of assays performed on solutions containing 100 mg / dl glucose and varying amounts of ascorbic acid were compared to these normalized responses.

Overall, the data presented in Table 2 show that test strips incorporating the composition of the present invention (Example 1) were less than 30 mg / dl, even in the presence of 200 mg / dl ascorbic acid. Figure 4 shows a detectable color change with high apparent glucose concentration. Thus, test strips containing the composition without metal ion complex and bromate ion (Example 2) show an apparent concentration of glucose of 0 mg / dl in the presence of 100 mg / dl ascorbic acid.

In particular, the test sample containing no ascorbate as an interferent (test sample A) showed glucose (100 mg / d) by the composition of Example 1 or the composition of Example 2.
l) is tested accurately. However, if the test sample contains ascorbate, the composition of Example 1 shows a response sufficient to indicate that greater than 30 mg / dl glucose is present in the test sample, with the presence of interfering ascorbate present. Although shown regardless of the amount of the acid salt, the test strip incorporating the composition of Example 2 contains 100 mg / dl or 200 mg / dl ascorbic acid in the test sample (test sample C and D), the test strips did not undergo a color change. In contrast, test strips incorporating the composition of the present invention still showed a glucose response greater than 30 mg / dl even when 200 mg / dl ascorbic acid was present in the test sample (test sample D). . Similarly, the composition of the present invention comprises 50 mg / d
l The presence of ascorbic acid in the test sample also resulted in improved assay results.

To further demonstrate the utility of the present invention, bromate ions and Fe (III) -HEDTA were found to effectively eliminate ascorbate interference in assays for cholesterol. Table 3 summarizes the results of the cholesterol assay in the five wet phases. To determine the effectiveness of a composition comprising cholesterol oxidase, peroxidase, tetramethylbenzidine (TMB) dye, metal ion complex, and co-oxidant to reduce or eliminate ascorbic acid interference in assays for cholesterol An assay was performed.

TABLE 3 Improved ascorbate resistance in assays for cholesterol ─ Test Cholesterol Peroxy Sample Water ¹⁾ Buffer ²⁾ Oxidase ³⁾ Dase ⁴⁾ TMB ⁵⁾ ──────────────────────────── Ａ A 0.86 0.10 10 10 10 B 0.85 0.10 10 10 10 C 0.84 0.10 10 10 10 D 0.83 0.10 10 10 10 E 0.82 0.10 10 10 10 ────────────── ──────────────────── Test Ascor Fe (III) -Cholester Color sample Binic acid ⁶⁾ HEDTA ⁷⁾ KBrO ₃ ⁸⁾ Roll ⁹⁾ Forming ¹⁰⁾ ─── Ａ A---10 Yes B 10--10 No C 10 10-10 No D 10 10 10 10 No E 10 10 20 10 Yes ────────── ──────────────────────── 1) Unit: milliliter (ml), each sample has a total volume of 1 ml
Met. 2) Unit: ml, buffer is pH 6.5, 1 mole of 2- (N-morpholino) ethanesulfonic acid (MES). 3) Units: GDS Technology, Inc. (Elkhart, Ind.) For 100 mg of cholesterol oxidase per microliter (μl) and 1 ml of water (1,330 units / ml)
More available. 4) Unit: μl, 100m peroxidase per 1ml of water
About g (103 units / mg). 5) Unit: μl, tetramethylbenzidine (TM) as an indicator dye dissolved in 1-methoxy-2-propanol
B) For a 100 mM solution. 6) Unit: μl, 0.56 M solution of ascorbic acid (10% by weight of ascorbic acid), test samples B to E each 5.6 mM
(Mmol) ascorbic acid. 7) Unit: μl, for a 100 mM solution of Fe-HEDTA, test samples CE were each 1.0 mM Fe (III)-.
Contains HEDTA. 8) Unit: μl, for a 0.2 M aqueous solution of potassium bromate, test sample D was 2.0 mM KBrO ₃ , test sample E was 4.0
Each contains mM KBrO ₃ . 9) Units: μl, serum containing 333 mg / dl (milligram / deciliter) of cholesterol, 10) Test samples were examined for blue color formation.

The tests shown in Table 3 were performed in 12 mm (millimeter) × 75 mm glass test tubes. Test sample A
For, cholesterol oxidase, peroxidase, TMB, and MES buffer were initially added to the tubes. The reaction was then initiated by adding serum containing cholesterol to the test tube. The total volume in the test tube is 1. for test sample A and test samples B to E.
It was 00 ml. After about 10 to 15 seconds, the reaction solution turned blue in color, indicating the presence of cholesterol in the test sample. Test sample A was a control and contained no ascorbic acid as a buffer, so the indicator dye underwent a sufficient color change in response to the amount of cholesterol present in test sample A.

Test sample B was subjected to the same assay, except that the concentration of ascorbic acid was 5.6 mM. Ascorbic acid interfered enough that no color change of the reaction solution to blue was observed. Test sample C was subjected to the same test as test sample B, except that Fe (III) -HEDTA was also 1.0 mM. Again, no color change to blue was observed. Although Fe (III) -HEDTA eliminated ascorbate interference, the production of ferrous ions from the interaction of Fe (III) -HEDTA with ascorbate resulted in secondary interference, where The ferrous ion interacted with the colored form of oxidized TMB to regenerate the ferric ion and the colorless form of reduced TMB. As a result, the test was a false negative test for cholesterol.

Test sample D was also tested for potassium bromate
Test sample D was subjected to the same assay as test sample C, except that mM was used. Again, no color change to blue was observed. However, in Test Sample E, which was the same as Test Sample D except that the potassium bromate was 4 mM, a color change to blue was observed. This color change is essentially the same as the test sample A in the degree and intensity of the color change, and thus the interference of ascorbic acid requires the metal ion complex and a sufficient amount of a suitable co-oxidant in the indicator reagent composition. It is shown that it can be overcome by the inclusion.

The use of other metal complexes and other co-oxidants in the indicator reagent composition of the present invention can also reduce or eliminate the interference effect of ascorbic acid in assays for a given analyte. To prove this, several dry phase test strips containing different metal ion complexes and different co-oxidants were prepared and used for glucose assays. For the test strip, filter paper was used as a carrier substrate, and an indicator reagent composition containing tetramethylbenzidine dye (TMB), glucose oxidase and peroxidase was incorporated into the carrier substrate. Further, the indicator reagent composition contained either a buffer, a co-oxidant or a metal ion complex, or a combination of a co-oxidant and a metal ion complex. Using this test strip, samples containing glucose as the predetermined analyte and ascorbic acid as the interferent were assayed. The test strips were then visually compared to determine the degree and intensity of the color change. Table 4 shows the results of the experiment.

Table 4 Metal complexes and co-oxidants that eliminate ascorbate interference ───── Experiment No.Buffer Metal complex Co-oxidant ──────────────────────────────────── 1 MES ⁴⁾ -0- -0- 2 MES -0- -0- 3 MES Fe (III) -HEDTA ⁵⁾ (5mM) -0- 4 MES -0- Bromate (20mM) 5 MES Fe (III ) -HEDTA (5mM) Bromate (20mM) 6 MES Fe (III) -HEDTA (5mM) Bromate (20mM) 7 MES -0- -0- 8 MES Fe (III) -HEDTA (5mM) Bromate Salt (20mM) 9 MES HAC ⁶⁾ (5mM) Bromate (20mM) 10 MES CPD ⁷⁾ (5mM) Bromate (20mM) 11 MES Pb (Ac) ₄ ⁸⁾ (25mM) Bromate (25mM) 12 MES Hg ^{+2 9)} (25mM) Bromate (25mM) 13 MES Hg ⁺² (25mM) and Fe (III) -HEDTA (5mM) -0- 14 MES Hg ⁺² (25mM) -0-15 ¹⁰⁾ Fe (III) -HEDTA (5mM) Chlorate (10mM) 16 Citrate Fe (III) -HEDTA (5mM) Chlorate (20mM) 17 Citrate Fe (III)- HEDTA (5mM) Chlorate (30mM) 18 Citrate -0- Chlorate (30mM) 19 Citrate Fe (III) -HEDTA (5mM) Perchlorate (10mM) 20 Citrate Fe (III ) -HEDTA (5mM) Perchlorate (20mM) 21 Citrate Fe (III) -HEDTA (5mM) Perchlorate (30mM) 22 Citrate -0- Perchlorate (30mM) 23 Citric acid Salt Fe (III) -HEDTA (5mM) Iodate (10mM) 24 Citrate Fe (III) -HEDTA (5mM) Iodate (20mM) 25 Citrate Fe (III) -HEDTA (5mM) Iodic acid Salt (30 mM) 26 Citrate -0- Iodate (30 mM) 27 PO ₄ ¹¹⁾ Gan-His-Cu ¹²⁾ Bromate (5 mM) 28 PO ₄ Gan-His-Cu -0- 29 PO ₄ Fe (III) -HEDTA (5mM) Chromate (5mM) 30 PO ₄ -0- Chromate (5mM) 31 PO ₄ Fe (III) -HEDTA (5mM) DBDH ¹³⁾ (5mM) 32 PO ₄ -0- DBDH (5mM) ──────────────────────────────────── Experimental strip number Glucose ¹⁾ Ascorbic acid ^{2 )} reactive ³⁾ ──────────────────── ─────────────── 1 100 100-2 100 50 + 3 100 100-4 100 100-5 -0-100-6 100 -0- ++++ 7 100 -0 -++++ 8 100 100 ++++ 9 100 100-10 100 50-11 100 50 + 12 100 50 ++++ 13 100 50 +++ 14 100 50 +++ 15 100 100 ++ 16 100 100 ++ 17 100 100 ++ 18 100 100-19 100 100 ++ 20 100 100 ++ 21 100 100 ++ 22 100 100-23 100 100 ++++ 24 100 100 ++++ 25 100 100 ++ ++ 26 100 100 ++++ 27 100 100 +++ 28 100 100-29 100 100 ++++ 30 100 100 + 31 100 100 ++++ 32 100 100 + ───────── ─────────────────────────── 1) Concentration of glucose in test sample, unit: mg / dl. 2) Concentration of ascorbic acid in test sample, unit: mg / dl
. 3) ++++ means that the reactivity of the test strip is essentially equal to the reactivity observed when immersed in a control sample without ascorbic acid, +++ means ++++ Slightly less reactive than ++, less reactive than +++, +
Inferior to ++ or-means no reactivity, respectively. 4) MES is 2- (N-morpholino) ethanesulfonic acid, present at 200 mM, resulting in a pH of 6.5. 5) Solution of ferric ion complex with HEDTA. 6) HAC is hexaamminecobalt (III) chloride. 7) CPD is cobalt (III) 2,4-pentanedionate. 8) Pb (Ac) ₄ is lead tetraacetate. 9) Hg ⁺² is mercuric nitrate solution. 10) Citrate is a citrate buffer, present at 400 mM, resulting in a pH of 6.5. 11) PO ₄ is a phosphate buffer, present at 400 mM, resulting in a pH of 6.5. 12) Gan-His-Cu is disclosed in U.S. Patent Application No. 337,62.
GANTREZ-histamine-copper complex disclosed in No. 0. 13) DBDH is diisopropylbenzene dihydroperoxide.

Table 4 shows that Fe (III) -HEDTA as a metal complex and potassium bromate as a co-oxidant are particularly useful in the indicator reagent composition of the present invention. Experiments 1 and 3 to 5 show that if either or both the metal ion complex and the co-oxidant are not present in the indicator reagent composition, the strip has no reactivity in the presence of 100 mg / dl ascorbate. (No discoloration). However, Experiment 2 showed that when the concentration of ascorbic acid in the test sample was reduced to 50 mg / dl, the strip showed little reactivity in the absence of the metal ion complex and co-oxidant. Experiment 7 is a control experiment which showed the reactivity of the strip to glucose (maximum discoloration) in the absence of ascorbic acid, co-oxidant and metal ion complex. The discoloration shown in Experiment 7 was used as a standard. The discoloration of the test strip in other experiments was compared to the standard discoloration of Experiment 7. Experiment 6 demonstrated that in the absence of ascorbate in the test sample, the metal ion complex and the co-oxidant did not adversely affect the reactivity of the test strip. Experiment 8 shows that the metal ion complex and the co-oxidant effectively overcome the interference effect of ascorbic acid,
This resulted in essentially the same strip activity as control experiment 7.

Experiments 9-11 indicated that cobalt ion complexes and lead ion complexes were not effective metal ion complexes in the indicator reagent compositions of the present invention. The result is
By comparing the reactivity of the test strips of experiments 9-11 with the reactivity of the test strips of experiments 8 and 2,
Most easily established, the test strip containing HAC / bromate (Experiment 9) lacks reactivity,
The Fe (III) -HEDTA / bromate containing test strip (Experiment 8) was fully reactive, and the CPD / bromate containing test strip (Experiment 10) showed that the metal ion complex and co-oxidant Less reactive than test strips not present (Experiment 2). Similarly, the lead ion complex and co-oxidant contained in the test strip of Experiment 11 could increase the reactivity of the test strip more than the reactivity of the test strip in Experiment 2 in the absence of the metal ion complex and co-oxidant. Did not.

Experiments 12 to 14 showed that mercuric ion was an effective metal ion for eliminating ascorbate interference in the indicator reagent composition of the present invention.
Experiment 14 showed that mercuric ion alone provided a relatively highly reactive test strip in the presence of ascorbic acid. Fe (III) with mercuric ion
) -HEDTA included in test strip (Experiment 1)
3) did not appreciably increase the reactivity of the test strip, but including bromate ion as a co-oxidant in the test strip along with mercuric ion improves the reactivity of the test strip. And resulted in the largest color change (Experiment 12).

Experiments 15-18 indicated that chlorate was an effective co-oxidant in the indicator reagent composition of the present invention. Experiment 3 shows that the inclusion of Fe (III) -HEDTA in the test strip without a co-oxidant was 100 mM.
In the presence of ascorbic acid in the test strips. Experiments 15-17
Test strips containing Fe (III) -HEDTA and chlorate ions showed an appreciably improved reactivity to test samples containing ascorbic acid as compared to the test strips of Experiment 3. Experiment 18 further showed that chlorate ions were not primary oxidants to ascorbic acid, but co-oxidants. Fe (III) -HEDTA
In the absence of
Showed no reactivity in the presence of ascorbic acid. Therefore, Fe (III) -HEDTA exists to eliminate primary interference of ascorbic acid, and chlorate ion exists to reduce the secondary interference effect of ferrous ion.

Similarly, Experiments 19-22 showed that perchlorate was an effective co-oxidant in the indicator reagent composition of the present invention. Similar to the above experiments involving chlorate ions, experiments 19-21 were performed using Fe (III) -HEDT.
Test strips containing A and perchlorate ions have been demonstrated to exhibit substantially improved reactivity to test samples containing ascorbic acid as compared to the test strips of Experiment 3. Experiment 22 showed that chlorate was a co-oxidant and not a primary oxidant of ascorbic acid. Perchlorate ion is Fe (III) -HEDT
In the absence of A, it does not improve the reactivity of the test strip, and therefore the perchlorate ion reduces the secondary interference by the oxidized form of the metal ion in the metal ion complex.

In contrast to chlorate and perchlorate, iodate is the primary oxidant, as shown in Experiments 23-26. Experiment 26 shows that iodate alone eliminates the interfering effects of ascorbate in the test sample, thus forming the test strip with maximum reactivity. Therefore, the presence of Fe (III) -HEDTA is unnecessary when iodate ions are present.

Experiments 27 and 28 showed that bromate was also an effective co-oxidant in indicator reagent compositions containing cupric ion complexes. The specific cupric ion complex tested is described in the above-cited US patent application Ser.
No. 620. However, other cupric ions are also bromate, chlorate, perchlorate, chromate, and organic oxidants such as peroxides, hydroperoxides or N-halogen compounds, and combinations thereof. It is envisaged that the indicator reagent composition of the present invention can be contained together with a co-oxidizing agent selected from the group consisting of combining.

Experiments 29 and 30 showed that chromate ions were also effective co-oxidants in compositions containing Fe (III) -HEDTA. Similarly, Experiment 3
1 and 32 showed that an organic oxidant similar to the hydroperoxide DBDH was also an effective co-oxidant in compositions containing Fe (III) -HEDTA.

[0155]

In conclusion, and according to an important feature of the invention, the ongoing and substantial problem of ascorbic acid interference in dry phase test strips for a given analyte is essentially eliminated, A more sensitive assay is provided, especially for low concentrations of a given analyte. The indicator reagent composition of the present invention essentially eliminates the primary interference of ascorbic acid by containing a metal ion complex. The indicator reagent composition also comprises bromate, chlorate, perchlorate, chromate, and organic oxidants such as peroxides, hydroperoxides or N-halogen compounds, and combinations thereof. By containing a co-oxidizing agent selected from the group, the secondary interference caused by the reduced form of the metal ion in the metal ion complex is also essentially eliminated. Essential elimination of primary and secondary interferences of ascorbic acid can be achieved by assays with dry phase test strips for a given analyte, such as interacting with occult blood, cholesterol or glucose, and oxidase, or peroxidase. It is an unexpected improvement in the art of assays for similar analytes that can either exhibit activity.

As described above, and according to an important feature of the present invention, by using the indicator reagent composition of the present invention,
More accurate and reliable assays can be performed for certain analytes in urine, serum, plasma and other test samples. In general, metal ion complexes and bromate, chlorate, perchlorate, chromate, and organic oxidants such as peroxides, hydroperoxides or N-halogen compounds, and combinations thereof The indicator reagent composition of the present invention, comprising a co-oxidant selected from the group consisting of: an improved primary interference of ascorbic acid and a secondary interference of the reduced form of the metal ion in the metal ion complex. Exhibit a better color change in response to the concentration of a given analyte in the test sample,
Does not interfere with the oxidation of the indicator dye by a given analyte and redox mediator and does not interfere with the ascorbate resistance provided by the metal ion complex.

Obviously, many modifications and variations of the present invention described above may be made without departing from the spirit and scope thereof, and are thus only subject to the limitations specified by the claims. Should be.

[Brief description of the drawings]

FIG. 1: 25 μM (micromolar) Fe (III) -H
Potassium bromate (KBr) at pH 6.5 in the presence of EDTA
KBrO monitored by a decreasing ascorbic acid peak at 265 nm (nanometers) when the concentration range of O ₃ ) was varied from 0 mM (mmol) to 10 mM.
₃ shows increased rate of ascorbic acid oxidation by 26
5 shows a series of plots of absorbance at 5 nm (A ₂₆₅ ) versus time (seconds).

FIG. 2: Rate constants (k,) for ascorbic acid oxidation in the presence of 25 μM Fe (III) -HEDTA and at pH 6.5.
2 is a graph in which sec ^-1 ) is plotted against potassium bromate concentrations ranging from 0 to 10 mM.

FIG. 3: pH 6.5 with 0.5 mM potassium bromate and Fe (III) -HEDTA ranging from 0 to 50 μM.
5 shows a series of plots of A ²⁶⁵ against time showing the rate of ascorbic acid oxidation at

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-2-286099 (JP, A) JP-A-56-63262 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/00-1/66 G01N 31/22

Claims (10)

(57) [Claims] 1. A method according to claim 1, wherein said analyte is capable of interacting with an oxidase or exhibiting peroxidase activity, wherein said analyte is present in said test sample upon contact with said test sample. A composition that can exhibit a color change sufficient to indicate the presence or concentration of a given analyte of: (a) an indicator dye, (b) a redox mediator, (c) a ferric ion, Mercuric ion, stannic ion, nickel (II) ion, manganese (III) ion,
Cadmium (II) ion, zinc (II) ion, molybdenum (V) ion, chromium (IV) ion, vanadium (II
A metal ion complex containing a metal ion selected from the group consisting of I) ions, cerium (IV) ions and combinations thereof; (d) bromate ions, chlorate ions, perchlorate ions,
Chromate ion, peroxide, hydroperoxide, N-
A composition comprising a co-oxidizing agent selected from the group consisting of halogen compounds and combinations thereof, and (e) a suitable carrier vehicle. 2. The composition of claim 1, wherein the indicator dye is present in an amount ranging from about 5 to about 60 millimoles per liter of the composition. 3. The composition according to claim 1, wherein the indicator dye is 3,3 ', 5,5'-tetramethylbenzidine. 4. The composition of claim 1, wherein the metal ion complex is present in an amount ranging from about 0.5 to about 50 millimoles per liter of the composition. 5. The method according to claim 5, wherein said analyte is capable of interacting with an oxidase or exhibiting peroxidase activity, wherein said analyte is present in said test sample upon contact with said test sample. A composition capable of exhibiting a color change sufficient to indicate the presence or concentration of a given analyte of: (a) an indicator dye, (b) a redox mediator, (c) mercuric nitrate, (D) bromate ion, chlorate ion, perchlorate ion,
Chromate ion, peroxide, hydroperoxide, N-
A composition comprising a co-oxidizing agent selected from the group consisting of halogen compounds and combinations thereof, and (e) a suitable carrier vehicle. 6. A method according to claim 1, wherein said analyte is capable of interacting with an oxidase or exhibiting peroxidase activity. A method for measuring the presence or concentration of: (a) using the test sample as an indicator dye; a redox mediator; ferric ion, mercury ion, stannic ion, nickel (II) ion, manganese (III) ) Ions, cadmium (I
I) ion, zinc (II) ion, molybdenum (V) ion, chromium (IV) ion, vanadium (III) ion,
A metal ion complex containing a metal ion selected from the group consisting of cerium (IV) ions and combinations thereof; bromate ions, chlorate ions, perchlorate ions, chromate ions, peroxides, hydroperoxides, Contacting a composition comprising a co-oxidant selected from the group consisting of N-halogen compounds and combinations thereof and a suitable carrier vehicle; (b) examining the composition from the intensity and degree of discoloration Determining a presence or concentration of said predetermined analyte in a sample. 7. A method for measuring the presence or concentration of occult blood in a biological fluid, the method comprising: (a) using the biological fluid as an indicator dye; a hydroperoxide;
Ferric ion, mercuric ion, stannic ion, nickel (II) ion, manganese (III) ion, cadmium (II) ion, zinc (II) ion, molybdenum (V)
Metal ion complex containing a metal ion selected from the group consisting of ion, chromium (IV) ion, vanadium (III) ion, cerium (IV) ion and a combination thereof; bromate ion, chlorate ion, perchlorine Contacting a composition comprising a co-oxidant selected from the group consisting of acid ions, chromate ions, N-halogen compounds and combinations thereof and a suitable carrier vehicle; (b) discoloration of the composition Measuring the presence or concentration of occult blood in the biological fluid from the intensity and degree of the occult blood. 8. A method for measuring the presence or concentration of glucose in a biological fluid, comprising: (a) using the indicator dye; glucose oxidase;
Peroxidase; ferric ion, mercuric ion, stannic ion, nickel (II) ion, manganese (II)
Selected from the group consisting of I) ions, cadmium (II) ions, zinc (II) ions, molybdenum (V) ions, chromium (IV) ions, vanadium (III) ions, cerium (IV) ions, and combinations thereof. A metal ion complex containing a metal ion; and a metal ion complex selected from the group consisting of bromate ion, chlorate ion, perchlorate ion, chromate ion, peroxide, hydroperoxide, N-halogen compound, and combinations thereof. Contacting a composition comprising an oxidizing agent and a suitable carrier vehicle; and (b) measuring the presence or concentration of glucose in the biological fluid from the intensity and degree of discoloration of the composition. Method. 9. A method for determining the presence or concentration of a predetermined analyte in a liquid sample, said method being capable of interacting with an oxidase or exhibiting peroxidase activity, (A) separating the liquid sample with an indicator dye, a redox mediator,
Ferric ion, mercuric ion, stannic ion, nickel (II) ion, manganese (III) ion, cadmium (II) ion, zinc (II) ion, molybdenum (V)
Ion, chromium (IV) ion, vanadium (III) ion, cerium (IV) ion and a metal ion complex containing a metal ion selected from the group consisting of a combination thereof, bromate ion, chlorate ion, perchlorine Reagent test containing a composition comprising a co-oxidant selected from the group consisting of acid ions, chromate ions, peroxides, hydroperoxides, N-halogen compounds and combinations thereof, and a suitable carrier vehicle Contacting with an analyte test device having a pad; and (b) examining the analyte test device for a color change in response to the content of the predetermined analyte present in the liquid sample. And how. 10. An analyte for determining the presence or concentration of a given analyte in a liquid sample, wherein said analyte is capable of interacting with an oxidase or exhibiting peroxidase activity. A support strip, a reagent test pad, and (a) an indicator dye, (b) a redox mediator, (c) ferric ion, mercuric ion, stannic ion, nickel (II) Ion, manganese (III) ion,
Cadmium (II) ion, zinc (II) ion, molybdenum (V) ion, chromium (IV) ion, vanadium (II
A metal ion complex containing a metal ion selected from the group consisting of I) ions, cerium (IV) ions and combinations thereof; (d) bromate ions, chlorate ions, perchlorate ions,
Chromate ion, peroxide, hydroperoxide, N-
A test comprising a co-oxidant selected from the group consisting of halogen compounds and combinations thereof; and (e) a composition comprising a suitable carrier vehicle and incorporated in said reagent test pad. Utensils.